Determination of fetal dna fraction in a sample

ABSTRACT

The present invention provides detection systems and methods for detection of loci and genomic regions in a sample, including mixed samples, using hybridization to an array.

CROSS-REFERENCE TO RELATED APPLICATIONS

The present application is a continuation of U.S. Ser. No. 14/453,396,filed 6 Aug. 2014, which is a continuation of U.S. Ser. No. 14/450,144filed 1 Aug. 2014, which is a continuation-in-part of U.S. Ser. No.13/013,732, filed 25 Jan. 2011, U.S. Ser. No. 13/205,490, filed 8 Aug.2011, and U.S. Ser. No. 13/205,603, filed 8 Aug. 2011, all of whichclaim priority to U.S. Ser. No. 61/371,605, filed 6 Aug. 2010; U.S. Ser.No. 13/205,490 is a continuation-in-part of U.S. Ser. No. 13/013,732,filed 25 Jan. 2011; and U.S. Ser. No. 13/205,603 is acontinuation-in-part of Ser. No. 13/013,732; U.S. Ser. No. 14/450,144 isa continuation-in-part of U.S. Ser. No. 13/316,154, filed 9 Dec. 2011,which claims priority to U.S. Ser. No. 61/436,135, filed 25 Jan. 2011,all of which are incorporated by reference in their entireties.

FIELD OF THE INVENTION

This invention relates to detection of target genomic regions fromsamples.

BACKGROUND OF THE INVENTION

In the following discussion certain articles and methods will bedescribed for background and introductory purposes. Nothing containedherein is to be construed as an “admission” of prior art. Applicantexpressly reserves the right to demonstrate, where appropriate, that thearticles and methods referenced herein do not constitute prior art underthe applicable statutory provisions.

Genetic abnormalities account for a wide number of pathologies,including pathologies caused by chromosomal aneuploidy (e.g., Downsyndrome), germline mutations in specific genes (e.g., sickle cellanemia), and pathologies caused by somatic mutations (e.g., cancer).Diagnostic methods for determining genetic anomalies have becomestandard techniques for identifying specific diseases and disorders, aswell as providing valuable information on disease source and treatmentoptions.

Copy number variations (CNVs) are alterations of genomic DNA thatcorrespond to specific regions of the genome-including entirechromosomes—that have been deleted or duplicated. CNVs can be caused bygenomic rearrangements such as deletions, duplications, inversions, andtranslocations. CNVs have been associated with various forms of cancer(Cappuzzo F, Hirsch, et al. (2005) J Natl Cancer Inst., 97(9):643-55),neurological disorders, including autism (Sebat, J., et al. (2007)Science 316(5823):445-9), and schizophrenia (St. Clair, D., (2008).Schizophr Bull 35(1):9-12).

Therefore, there is a need for methods of screening for copy numbervariations that employs an efficient, reproducible assay and detectionsystem.

SUMMARY OF THE INVENTION

This Summary is provided to introduce a selection of concepts in asimplified form that are further described below in the DetailedDescription. This Summary is not intended to identify key or essentialfeatures of the claimed subject matter, nor is it intended to be used tolimit the scope of the claimed subject matter. Other features, details,utilities, and advantages of the claimed subject matter will be apparentfrom the following written Detailed Description including those aspectsillustrated in the accompanying drawings and defined in the appendedclaims.

The present invention provides methods for detecting geneticcharacteristics in a sample, including copy number variations (CNVs),insertions, deletions, translocations, polymorphisms and mutations. Theinvention employs the technique of interrogating loci from two or moretarget genomic regions using at least two fixed sequenceoligonucleotides for each interrogated locus, and joining the fixedsequence oligonucleotides either directly or indirectly via ligation.The ligation products from different loci in a selected genomic regionscomprise nucleic acid capture regions designed to include a regioncomplementary to one or more capture probes on a solid support. Thecapture region comprises one or more detectable labels that identify aligation product as originating from a specific target genomic region.Identification of ligation products from different target genomicregions is achieved by binding of the capture regions of the ligationproducts to complementary capture probes on the solid support.

In a specific embodiment, the invention provides an assay method forproviding a statistical likelihood of a fetal aneuploidy comprisingproviding a maternal sample comprising maternal and fetal cell free DNA,interrogating one or more loci from a first target genomic region usingsequence-specific oligonucleotides that comprise a capture region,interrogating one or more loci from a second target genomic region usingsequence-specific oligonucleotides that comprise a capture region,detecting the isolated selected loci from the first and second targetgenomic regions via hybridization to an array, quantifying total countsof the isolated loci to determine a relative frequency of first andsecond target genomic regions, interrogating selected polymorphic locifrom at least one target genomic region different from the first andsecond target genomic regions using sequence-specific oligonucleotides,detecting isolated selected polymorphic loci, quantifying total countsof the isolated selected polymorphic loci to calculate a percentage ofthe fetal cell free DNA in the maternal sample, calculating astatistical likelihood a fetal aneuploidy in the maternal sample,wherein the relative frequency of loci from the first target genomicregion, the relative frequency of loci from the second target genomicregion, and quantified counts from the isolated selected polymorphicloci to provide a statistical likelihood of the presence of a fetalaneuploidy.

In other specific embodiments, the invention provides an assay methoddetermining the presence or absence of a fetal aneuploidy comprisingproviding a maternal sample comprising maternal and fetal cell free DNA,interrogating one or more loci from a first target genomic region usingsequence-specific oligonucleotides that comprise a capture region,interrogating one or more loci from a second target genomic region usingsequence-specific oligonucleotides that comprise a capture region,detecting the loci from the first and second target genomic regions viahybridization to an array, quantifying total counts of the loci todetermine a relative frequency of first and second target genomicregions, and determining the presence or absence of a fetal aneuploidyin the maternal sample based on a deviation from expected counts of theisolated loci using the relative frequency of loci from the first targetgenomic region and the relative frequency of loci from the second targetgenomic region. In certain aspects, the deviation from expected countsis determined using a threshold level determined from a representativepopulation of samples, and preferably a representative populationcomprising samples from patients of similar maternal age and/orgestational age.

In specific aspects, the interrogation of the loci from the first andsecond genomic regions uses hybridization followed by ligation. In morespecific aspects, an amplification step is performed after thehybridization and ligation steps. In other specific aspects, theamplification is universal amplification using the polymerase chainreaction.

In preferred aspects, the ligation products from two or more differentgenomic regions are identified using a single solid support with captureprobes; e.g., an array comprising capture probes complementary tomultiple capture regions indicative of the different target genomicregions. Upon introducing a pool of ligation products originating fromtwo or more different genomic regions to the array, ligation productshaving the same capture region will competitively hybridize tocomplementary capture probes on the array, and the relative frequency ofligation products from each genomic region can be estimated based on theamount of detected label bound to the capture probes. In this manner,the relative frequencies of the target genomic regions themselves may bedetermined. The relative frequencies of each target genomic region maybe determined by identifying the binding of capture regions on theligation products corresponding to each selected locus from each targetgenomic region to specific, known locations on the array, or byestimating total fluorescence from the array following binding of theligation products originating from the target genomic regions.

In certain preferred embodiments, the capture regions and capture probesdo not reflect the specific target genomic region nucleotide sequence,and are instead “engineered” sequences that serve as surrogates toidentify specific target genomic regions; thus, the nucleotide sequenceof the ligation product corresponding to the target genomic region doesnot need to be determined directly. The use of the capture regions onthe ligation products allows the binding of the ligation products to thecapture probes on the array to indicate the larger target genomic regionfrom which the ligation product originates without the need to sequencethe portion of the ligation product corresponding to the actualnucleotide sequence of the target genomic region. Because the captureregions and capture probes are engineered sequences, they can be thoughtof as “universal” sequences; that is, these capture regions and captureprobes can be used in conjunction with any number of different assays,the only difference being the target sequence(s) associated with thecapture region(s).

In one embodiment, the arrays of the present invention comprise captureprobes that all have substantially the same sequence. In anotherembodiment, the arrays used comprise two to several different featureswith capture probes having substantially the same sequence. These arraysare in contrast to arrays known in the art that identify individualsequences by complementarity to individual features with each featurecomprising a nucleic acid sequence different from the other features.The use of a single or a limited number of complementary capture probesequences in the individual features on an array can simplify thebiochemistry needed to create the array and reduce potential spuriousdifferences in detection frequency resulting, e.g., from differences inbinding affinity between the capture regions on the ligation productsand the capture probes.

In specific embodiments, the arrays comprise two or more differentcapture probes used to detect individual ligation products from two ormore different target genomic regions, two or more different loci from asingle target genomic region, or two or more different alleles from aselected locus. That is, capture probes of different sequence hybridizeto capture regions on ligation products that correspond to differenttarget genomic regions, different loci from a single target genomicregion, or different alleles from a selected locus. The capture regionson the ligation products are associated with labels indicative of thetarget genomic region or selected locus, or indicative of the alleles ofa polymorphism, from which the ligation product originated.

Thus, it is a preferred embodiment of the invention that the ligationproducts are identified using a capture probe that is complementary to acapture region introduced in or to the ligation product, but that doesnot identify the target genomic region to which the ligation productcorresponds solely by hybridization to a feature complementary to thetarget genomic region. In some embodiments, the capture probe is in partcomplementary to a target genomic region and in part complementary to acapture region in a ligation product. In a preferred embodiment, thecapture region used to identify a ligation product which corresponds toa target genomic region is not complementary to any portion of thetarget genomic region.

Preferably the capture region is introduced as part of one of the fixedsequence oligonucleotides prior to ligation, although the capture regionmay be attached (e.g., via ligation of an adaptor) to one or both endsof the ligation product of the fixed sequence oligonucleotides followingthe ligation procedure.

It is another feature of the hybridization assay format of the inventionthat quantification of target genomic regions when using capture regionscan be achieved by quantifying labels that are associated with theligation products from the loci within the target genomic region withoutactually determining the sequence of the ligation products correspondingto the target genomic regions. In this manner, the frequency of ligationproducts from a target genomic region (and thus the frequency of thetarget genomic regions themselves) can be estimated without the need todetect the actual nucleotide sequence of the loci from that targetgenomic region.

In many preferred embodiments, quantification of the labels bound to thecapture probes on the array is the only readout necessary to estimatethe levels or amounts of ligation products produced from each targetgenomic region, which in turn can be used to estimate the frequency ofthe target genomic regions.

It is an advantage of the methods of the invention that the ligationproducts can be associated with multiple different detectable labelsand/or capture regions. The use of different labels and/or captureregions in different experiments can mitigate any frequency bias fromthe use of a particular detectable label, capture region or captureprobe.

In certain embodiments, the present invention provides methods fordetecting frequencies of first and second target genomic regions in asample comprising: introducing a first set of first and second fixedsequence oligonucleotides to a sample under conditions that allow thefirst and second fixed sequence oligonucleotides to hybridizespecifically to complementary regions in loci from a first targetgenomic region, wherein at least one of the first or second fixedsequence oligonucleotide of the first set comprises a capture region anda first label; introducing a second set of first and second fixedsequence oligonucleotides to a sample under conditions that allow thefirst and second fixed sequence oligonucleotides to hybridizespecifically to complementary regions in loci from a second targetgenomic region, wherein at least one of the first or second fixedsequence oligonucleotide of the second set comprises a capture regionand a second label; ligating the hybridized fixed sequenceoligonucleotides to create ligation products complementary to the loci;introducing the ligation products to an array comprising capture probesunder conditions that allow the capture probes on the array to hybridizespecifically to the capture regions of the ligation products; detectingthe first and second labels; and quantifying a relative frequency of thefirst and second labels to quantify the relative frequency of the firstand second target genomic regions. In some aspects, the capture regionsof the first and second sets of ligation products are different. Inother aspects the capture regions of the first and second sets ofligation products are the same.

In a preferred aspect, the first and second capture regions of the firstand second sets of oligonucleotides are the same. In other aspects, asmall number of capture regions are used in the fixed sequenceoligonucleotides of both the first and the second set. In other aspects,the first capture regions corresponding to the first target genomicregion are different from the second capture regions corresponding tothe second genomic region.

In other preferred embodiments, in addition to interrogating first andsecond target genomic regions, polymorphic sequences comprising SNPsfrom two or more selected polymorphic loci also are interrogated.Selected polymorphic loci are interrogated using a third set of fixedsequence oligonucleotides to determine allele frequencies. The allelefrequencies are used, e.g., to calculate the percent of fetal nucleicacids present in a maternal serum sample.

In certain embodiments, the first and second fixed sequenceoligonucleotides do not hybridize adjacently to the loci in the targetgenomic regions, and instead have an intervening region or “gap” betweenthe fixed sequence oligonucleotides of a set hybridized to a locus. Thisintervening region may be filled, e.g., using a polymerase and dNTPs toextend the end of one fixed sequence oligonucleotide so that the endbecomes adjacent to the end of the other hybridized oligonucleotide ofthe set. In another embodiment, the intervening region may be filledusing one or more “gap-filling” or “bridging” oligonucleotides that bindbetween and adjacent to the fixed sequence oligonucleotides of a set. Inthe latter case, preferably the ligation step will ligate all of theoligonucleotides into a single, contiguous ligation product comprising asingle capture region which can then be detected on an array. In yetanother embodiment, a combination of bridging oligonucleotides and dNTPsand polymerase can be used to fill the intervening space between thefixed sequence oligonucleotides.

In the above-described embodiments, a set of fixed sequence nucleicacids is used which comprises two separate fixed sequenceoligonucleotides designed to hybridize to two separate regions in eachselected locus (either adjacently or non-adjacently). In someembodiments, however, a set of fixed sequence oligonucleotides cancomprise a single probe with regions at either end complementary to aselected locus. Upon hybridization of this single probe to a locus, theprobe forms a circular structure that may or may not be adjacentlyhybridized on the locus. Such “precircle” probes can also hybridize witha gap between the ends of the probe, which gap may be filled by thehybridization of one or more bridging oligonucleotides, by extension ofone end of the probe using polymerase and dNTPs, or a combinationthereof.

In certain aspects, the detectable labels are directly associated with(i.e., covalently or non-covalently bound to a capture region that is inone of the fixed sequence oligonucleotides of each set. In anotherembodiment, the ligation products are amplified following ligation,e.g., in a universal amplification, and the detectable label isassociated with a capture region contained within a primer used for theamplification. In other specific aspects, the isolated loci from thefirst and second target genomic regions and the ligation products fromthe selected polymorphic loci are amplified in a single vessel. In otheraspects, the detectable labels are covalently or non-covalently bound toan oligonucleotide that hybridizes to a complementary sequence on theligation products, amplicons or cleavage products thereof. Such labeledoligonucleotides may be hybridized to the ligation products prior to orafter introduction of the ligation products to the capture probe array.

In other embodiments of the invention, the capture regions on theligation products corresponding to different target genomic regionscomprise different sequences, and the comparative frequency of at leasta first and a second target genomic region are determined based on theuse of different detectable labels associated with the different captureregions.

In certain aspects, copy number variants are is detected by analteration of an expected ratio of bound detectable label from the boundligation products from the target genomic regions in the sample. Incertain specific aspects copy number variants are detected by anincreased or decreased level of hybridization of a first set of ligationproducts from a first selected locus as compared to a second set ofligation products from a second selected locus.

The relative frequency of loci in a sample can be used to determine notonly copy number variation for a small target genomic region, but alsoin conjunction with and/or in comparison to other loci, the relativefrequency of loci may be used to determine the copy number variation oflarger target genomic regions, including partial or whole chromosomes.

In another general aspect of the invention, a method for detectingfrequencies of first and second target genomic regions in a sample isprovided comprising introducing a first set of first and second fixedsequence oligonucleotides to a sample under conditions that allow thefirst and second fixed sequence oligonucleotides to hybridizespecifically to complementary regions in loci in the first targetgenomic region to create first hybridized fixed sequenceoligonucleotides; introducing a second set of first and second fixedsequence oligonucleotides to the sample under conditions that allow thefirst and second fixed sequence oligonucleotides to hybridizespecifically to complementary regions to create second hybridized fixedsequence oligonucleotides; and ligating the hybridized fixed sequenceoligonucleotides of each set to create ligation products complementaryto loci in the first and second target genomic regions. At least onefixed sequence oligonucleotide of each set comprises a capture regioncomprising a sequence complementary to capture probes on an array and abinding region for a detectable label. The ligation products areintroduced to the array comprising capture probes complementary to thecapture regions of the ligation products under conditions that allow thecapture probes to specifically hybridize to the capture regions of theligation products. A first detectable label is introduced to the arrayunder conditions that allow the detectable label to specificallyhybridize to the binding region of the capture region on the ligationproducts from the loci from the first target genomic region, and asecond detectable label is introduced to the array under conditions thatallow the detectable label to specifically hybridize to the bindingregions of the capture region on the ligation products from the locifrom the second target genomic region. The first and second detectablelabels are detected and quantified to provide a relative frequency ofthe first and second target genomic regions in the sample. As discussed,above, in relation to one embodiment, in addition to interrogating firstand second target genomic regions, aspects of this embodimentinterrogate polymorphic sequences comprising SNPs from two or moreselected polymorphic loci. Selected polymorphic loci are interrogatedusing a third set of fixed sequence oligonucleotides to determine allelefrequencies. The allele frequencies are used, e.g., to calculate thepercent of fetal nucleic acids present in a maternal serum sample. Alsoas discussed, above, in some aspects of this embodiment, “gap-filling”or “bridging” oligonucleotides may be employed in addition to the twofixed sequence oligonucleotides, and in some aspects of this embodiment,the fixed sequence oligonucleotides or the bridging oligonucleotides areallele-specific, as described in detail infra.

In specific aspects, the assay of the invention provides identifying lowfrequency alleles from the isolated selected polymorphic loci where thematernal DNA is homozygous and the non-maternal DNA is heterozygous,computing a sum of low frequency alleles from the isolated selectedpolymorphic loci, and calculating a statistical likelihood of a fetalaneuploidy in the maternal sample using the sum of the low frequencyalleles from the isolated selected polymorphic loci to calculatestatistically significant differences in target genomic regionfrequencies for the first and second target genomic regions, and whereina statistically significant difference in chromosomal frequency providesa statistical likelihood of the presence of a fetal aneuploidy.

In yet another general aspect of the invention, a method is provided fordetecting frequencies of first and second target genomic regions in asample, the method comprising providing a sample; introducing a firstset of first and second fixed sequence oligonucleotides to the sampleunder conditions that allow the fixed sequence oligonucleotides tohybridize specifically to complementary regions in first loci of thefirst target genomic region to create first hybridized fixed sequenceoligonucleotides; and introducing a second set of first and second fixedsequence oligonucleotides to the sample under conditions that allow thefixed sequence oligonucleotides to hybridize specifically tocomplementary regions in second loci of the second target genomic regionto create second hybridized fixed sequence oligonucleotides. At leastone of the fixed sequence oligonucleotides of the first set of fixedsequence oligonucleotides comprises a capture region and a label bindingregion complementary to a first detectable label, and at least one ofthe fixed sequence oligonucleotides of the second set of fixed sequenceoligonucleotides comprises substantially the same capture region as thefirst set and a label binding region complementary to a seconddetectable label. The fixed sequence oligonucleotides of the first andsecond set are ligated to create first and second ligation productscomprising regions complementary to the first and second loci,respectively, and labeled with a first and second detectable label. Thefirst and second ligation products are introduced to an array comprisingcapture probes under conditions that allow the capture probes tospecifically hybridize to the capture regions of the ligation products.The first and second labels are detected and quantified to determine arelative frequency of the first and second labels, thereby quantifying arelative frequency of the first and second target genomic regions in thesample.

The detectable labels may be introduced to the ligation products priorto the introduction of the ligation products to the array.Alternatively, the detectable labels may be introduced to the arrayfollowing hybridization of the ligation products.

Again, in certain embodiments, the methods further employ the extensionof at least one fixed sequence oligonucleotide hybridized to a sequenceof interest. That is, in some embodiments the fixed sequenceoligonucleotides that hybridize to one or more loci may not hybridizeadjacently, leaving a “gap” or “intervening” region. This interveningregion may be filled, e.g., using a polymerase and dNTPs to extend theend of one fixed sequence oligonucleotide so that the end is adjacent tothe end of the other hybridized fixed sequence oligonucleotide of theset. In another embodiment, the intervening region may be filled usingone or more “gap-filling” or “bridging” oligonucleotides that bindbetween and adjacent to the fixed sequence oligonucleotides of a set. Inthe latter case, preferably the ligation step will ligate all of theoligonucleotides into a single, contiguous ligation product comprising asingle capture region which can then be detected on an array. Also, acombination of bridging or gap-filling oligonucleotides and dNTPs andpolymerase can be used to fill the gap. Additionally in someembodiments, pre-circular, padlock or molecular inversion probes may beused in lieu of two fixed sequence oligonucleotides in a set.

When gap-filling or bridging oligonucleotides are used, the bridgingoligonucleotides typically are short, preferably between 2-30nucleotides and more preferably between 3-28 nucleotides in length. Inone aspect, the bridging oligonucleotides can be designed to providedegeneracy at multiple or all positions, e.g., the bridgingoligonucleotides may be full or partial randomers with various sequencevariations to ensure detection of the loci even if a locus contains apolymorphic nucleotide at one or more positions. The degeneracy of thebridging oligonucleotide can be designed based on the predictedpolymorphisms that may be present in the loci. Alternatively, in anotheraspect the pool of bridging oligonucleotides used in a reaction canprovide limited degeneracy targeting specifically one or more positionsbased on predicted polymorphisms that may be present in the regions ofthe loci. In yet another aspect, the pool of bridging oligonucleotidesused in a reaction can provide degeneracy for each internal position,with the nucleotides adjacent to the sites of ligation with the fixedsequence oligonucleotides remaining fixed. It is an advantage that usingdegenerate bridging oligonucleotides obviates the need to predeterminethe maternal and fetal polymorphic content for a selected locus prior toemploying the detection methods of the present invention.

In another aspect, the bridging oligonucleotide is longer than 10nucleotides in length and is preferably 18-30 nucleotides in length. Ina preferred aspect, there is a single bridging oligonucleotidecomplementary to each selected locus designed to hybridize between theregions of the selected locus complementary to the first and secondfixed sequence oligonucleotides. In another aspect, two or more bridgingoligonucleotides are designed to hybridize between the fixed sequenceoligonucleotides at each selected locus, and preferably the bridgingoligonucleotides hybridize adjacently to the first and second fixedsequence oligonucleotides.

In the situation where there are two bridging oligonucleotides, threeligation events occur per selected locus: ligation between the firstfixed oligonucleotide and the first bridging oligonucleotide, ligationbetween the first and second bridging oligonucleotides, and ligationbetween the second bridging oligonucleotide and the second fixedsequence oligonucleotide. In another aspect, there may be gaps betweenthe bridging oligonucleotides and/or between the bridgingoligonucleotides and the fixed sequence oligonucleotides. These gaps canbe filled by extension—e.g., by use of polymerase and dNTPs-prior toligation.

In one aspect of the invention, the first and second fixed sequenceoligonucleotides are introduced to the sample and specificallyhybridized to the complementary portions of the loci prior tointroducing the bridging oligonucleotides to the sample. In anotheraspect, the bridging oligonucleotides are introduced to the sample atthe same time the first and second sets of fixed sequenceoligonucleotides are introduced to the sample.

In another general aspect of the invention, a method for determining apresence or absence of an aneuploidy in a mixed sample is provided, themethod comprising providing a mixed sample; introducing a first set offirst and second fixed sequence oligonucleotides to the mixed sampleunder conditions that allow the fixed sequence oligonucleotides tohybridize specifically to first loci on a first chromosome to createfirst hybridized fixed sequence oligonucleotides, where the first set offixed sequence oligonucleotides comprises a first capture region and afirst label binding region; introducing a second set of first and secondfixed sequence oligonucleotides to the mixed sample under conditionsthat allow the fixed sequence oligonucleotides to hybridize specificallyto second loci on a second chromosome to create second hybridized fixedsequence oligonucleotides, where the second set of fixed sequenceoligonucleotides comprises a second capture region and a second labelbinding region; ligating the hybridized oligonucleotides to createligation products complementary to the loci; introducing the ligationproducts to an array comprising capture probes under conditions thatallow the capture probes to hybridize specifically to the first andsecond capture regions of the ligation products; introducing a firstlabeled oligonucleotide to the array under conditions that allow atarget recognition region of the first labeled oligonucleotide tohybridize specifically to the first label binding region; introducing asecond labeled oligonucleotide to the array under conditions that allowa target recognition region of the second labeled oligonucleotide tohybridize specifically to the second label binding region; detectingfirst and second labels; quantifying relative frequencies of the firstand second labels, thereby quantifying a relative frequency of the firstand second chromosome (or genomic region) in the mixed sample, wherein astatistically significant difference in the relative frequencies of thelabels on the array is indicative of the presence or absence of achromosomal aneuploidy in the mixed sample.

In alternative embodiments, a “threshold” level can be used to determinethe presence or absence of a fetal aneuploidy based on the observeddeviation of the relative frequency of the first and second chromosomein the mixed sample. This threshold may be determined, e.g., usingtechniques such as those disclosed in U.S. App. 2012/0149583,2013/0324420, 2013/0029852 and U.S. Pat. No. 8,532,936. In certainaspects, the deviation from expected counts is determined using athreshold level determined from a representative population of samples,and preferably a representative population comprising samples frompatients of similar characteristics, such as prior risk profile,maternal age and/or gestational age.

In preferred aspects of the invention, the sample DNA is bound to asolid support, either before, during or after the addition of the fixedsequence oligonucleotides. In preferred aspects of the invention, theassays employ steps to remove unhybridized oligonucleotides prior tocreation of ligation products, e.g., by washing or by exonucleasedigestion. In other preferred aspects, the ligation products areisolated following ligation but prior to further processing and/orintroduction to the array for detection. In other preferred embodiments,the ligation products are amplified, preferably using universal primers,to form amplicons. In other preferred embodiments, the amplicons aresubsequently cleaved to form cleaved amplicons before hybridization toan array. In embodiments involving cleavage of the ligation products,the cleaved region comprising the capture regions is preferablyseparated from the remainder of the cleavage products prior tointroduction of the capture region portion to the array.

In certain aspects, the sample DNA, ligations products and/or theamplification products are isolated using conventional techniques in theart. For example, the hybridization complexes (e.g., the fixed sequenceoligonucleotides bound to the target loci), ligations products and/orthe amplification products may be isolated by attachment to a solidsubstrate followed by a separation step, e.g. washing or nucleasedigestion. In specific examples, they may be isolated using attachmentto magnetic beads. In other specific examples, they may be isolatedusing attachment to a substrate with a binding partner, e.g. theoligonucleotide is biotinylated and the substrate comprises avidin orstreptavidin. In aspects in which precircle probes are used, thehybridization complexes and/or ligation products may be isolated bynuclease destruction of non-circularized probes.

In some aspects of this embodiment, the first and second capture regionshave the same nucleotide sequence. In other aspects of this embodiment,the first and second capture regions have different nucleotidesequences. Also as discussed, above, in relation to other embodiments,in addition to interrogating first and second target genomic regions,aspects of this embodiment interrogate polymorphic sequences comprisingSNPs from two or more selected polymorphic loci. Selected polymorphicloci are interrogated, e.g., using a third set of fixed sequenceoligonucleotides to determine allele frequencies. The allele frequenciesare used, e.g., to calculate the percent of fetal nucleic acids presentin a maternal serum sample.

As discussed above, in some aspects of this embodiment, extensionligation and/or “bridging” oligonucleotides may be employed in additionto the two fixed sequence oligonucleotides. Accordingly, the inventionprovides a method for determining a likelihood of a fetal aneuploidycomprising the steps of providing a maternal sample comprising maternaland fetal cell free DNA, introducing first sets of two fixed sequenceoligonucleotides complementary to loci in a first target genomic regionin the maternal sample under conditions that allow a complementaryregion of each fixed sequence oligonucleotide to specifically hybridizeto the loci, wherein at least one of the two fixed sequenceoligonucleotides of each set comprises a universal primer site and acapture region, introducing second sets of two fixed sequenceoligonucleotides complementary to loci in a second target genomic regionin the maternal sample under conditions that allow a complementaryregion of each fixed sequence oligonucleotide to specifically hybridizeto the loci, wherein at least one of the two fixed sequenceoligonucleotides of each set comprises a universal primer site and acapture region, introducing third sets of two fixed sequenceoligonucleotides complementary to a set of polymorphic loci in a targetgenomic region that is different from the first target genomic region inthe maternal sample under conditions that allow a complementary regionof each fixed sequence oligonucleotide to specifically hybridize toselected polymorphic loci, wherein at least one of the two fixedsequence oligonucleotides of each set comprises a universal primer siteand a capture region, introducing bridging oligonucleotides to thematernal sample under conditions that allow the bridgingoligonucleotides to specifically hybridize to complementary regions inthe loci between the fixed sequence oligonucleotides, ligating thehybridized first and second fixed sequence oligonucleotides and thebridging oligonucleotides to create ligation products complementary tothe loci, isolating the ligation products, amplifying the isolatedligation products using the universal primer sites, applying theamplified ligation products to an array, wherein the array comprisescapture probes complementary to the capture regions on the ligationproducts, quantifying a relative frequency of each allele from theselected polymorphic loci to determine a percent fetal cell-free DNA inthe sample, quantifying a relative frequency of loci from the firsttarget genomic region and a relative frequency of loci from the secondtarget genomic region, and computing a likelihood of the presence orabsence of a fetal aneuploidy using the relative frequency of the locifrom the first and second target genomic regions and the percent fetalcell-free DNA to determine the likelihood of the presence or absence ofa fetal aneuploidy.

The invention also provides a method for determining a likelihood of afetal aneuploidy comprising the steps of providing a maternal samplecomprising maternal and fetal cell free DNA, introducing first sets oftwo fixed sequence oligonucleotides complementary to loci in a firsttarget genomic region in the maternal sample under conditions that allowa complementary region of each fixed sequence oligonucleotide tospecifically hybridize to the loci, wherein at least one of the twofixed sequence oligonucleotides of each set comprises a universal primersite and a capture region, introducing second sets of two fixed sequenceoligonucleotides complementary to loci in a second target genomic regionin the maternal sample under conditions that allow a complementaryregion of each fixed sequence oligonucleotide to specifically hybridizeto the loci, wherein at least one of the two fixed sequenceoligonucleotides of each set comprises a universal primer site and acapture region, introducing third sets of two fixed sequenceoligonucleotides complementary to a set of polymorphic loci in a targetgenomic region that is different from the first target genomic region inthe maternal sample under conditions that allow a complementary regionof each fixed sequence oligonucleotide to specifically hybridize toselected polymorphic loci, wherein at least one of the two fixedsequence oligonucleotides of each set comprises a universal primer siteand a capture region, extending at least one of the hybridized fixedsequence oligonucleotides using dNTPs and a polymerase to createadjacently hybridized oligonucleotides, ligating the adjacentlyhybridized oligonucleotides to create ligation products complementary tothe loci, isolating the ligation products, amplifying the isolatedligation products using the universal primer sites, applying theamplified ligation products to an array, wherein the array comprisescapture probes complementary to the capture regions on the ligationproducts, quantifying a relative frequency of each allele from theselected polymorphic loci to determine a percent fetal cell-free DNA inthe sample, quantifying a relative frequency of loci from the firsttarget genomic region and a relative frequency of loci from the secondtarget genomic region, and computing a likelihood of the presence orabsence of a fetal aneuploidy using the relative frequency of the locifrom the first and second target genomic regions and the percent fetalcell-free DNA to determine the likelihood of the presence or absence ofa fetal aneuploidy.

In certain aspects, the invention further comprises comparing therelative frequency of the loci from the first and second target genomicregions and adjusting the relative frequency of the loci from the firstand second target genomic regions based on the percent fetal cell-freeDNA to determine the likelihood of the presence or absence of a fetalaneuploidy. In specific aspects, the relative frequencies of eachselected locus for each target genomic region are summed and the sumsfor each chromosome are compared to calculate a target genomic regionratio.

The percent fetal cell free DNA of a sample can be calculated bydetecting levels of one or more non-maternal contributed loci, e.g.,non-maternal loci on the Y-chromosome and/or non-maternal loci areautosomal loci. In preferred aspects, the non-maternal loci comprise oneor more genetic variations compared to maternal loci, e.g., SNPs ormethylation differences.

In certain embodiments, the ligation products are cleaved (e.g., usingenzymatic cleaving mechanisms such as a restriction endonuclease) toreduce the size of the ligation product while leaving the capture regionand label binding region available for detection. In certain aspects,the cleavage occurs after the universal amplification.

In preferred embodiments, the loci and fixed sequence oligonucleotideshybridized to the loci are isolated from unbound fixed sequenceoligonucleotides following hybridization to remove excess unboundoligonucleotides in the reaction; e.g., through a washing step orenzymatic degradation of the unbound oligonucleotides.

The first and second sets of fixed sequence oligonucleotides used in themethods preferably comprise—in addition to at least one captureregion-universal primer regions that may be used to amplify the ligationproducts. Alternatively, universal primer sequences may be added to theends of the ligation products following ligation, e.g., through theintroduction of adapters comprising universal primer sequences.

In certain aspects, the fixed sequence oligonucleotides of the inventioncomprise one or more indices. These indices may serve, in addition tothe capture regions, as surrogate sequences to identify the loci, or aparticular allele of a locus. In particular, these indices may serve assurrogate identification sequences to detect hybridization of theligation product or amplicons thereof to an array. In specific methods,the first or second fixed sequence oligonucleotide in each set of fixedsequence oligonucleotides comprises an allele index that associates aspecific allele with the fixed sequence oligonucleotide.

In certain specific aspects, the method is carried out for at least 50loci from each target genomic region, more preferably between 50-100loci, more preferably between 100-200 loci, more preferably between200-500 loci, more preferably between 500-1000 loci, preferably between1000-2000 loci, preferably between 2000-5000 loci, and preferablybetween 5000-10,000 loci from a target genomic region, or anyintervening range therein. In certain aspects, in addition to the targetgenomic regions, at least 50 selected polymorphic loci are interrogated.More preferably, between 50-100 selected polymorphic loci areinterrogated, more preferably between 100-200 selected polymorphic loci,more between 200-500 selected polymorphic loci, more between 500-1000selected polymorphic loci, between 1000-2000 selected polymorphic loci,between 2000-5000 selected polymorphic loci, and between 5000-10,000selected polymorphic loci are interrogated, including all interveningranges.

In other aspects, the assay methods are estimated to detect at least 5capture regions corresponding to each locus within a target genomicregion, more preferably at least 10 capture regions corresponding toeach locus within a target genomic region, more preferably at least 20capture regions corresponding to each locus within a target genomicregion, preferably at least 50 capture regions corresponding to eachlocus within a target genomic region, more preferably at least 100capture regions corresponding to each locus within a target genomicregion, more preferably at least 200 capture regions corresponding toeach locus within a target genomic region. In some embodiments, no morethan 5000 capture regions corresponding to each locus within a targetgenomic region are detected for each sample. In other embodiments nomore than 2000 capture regions corresponding to each locus within atarget genomic region are detected for each sample.

These aspects and other features and advantages of the invention aredescribed in more detail below.

BRIEF DESCRIPTION OF THE FIGURES

FIG. 1 is a flow chart describing one general aspect of the invention.

FIG. 2 illustrates an embodiment of a method of the invention thatutilizes hybridization detection of loci.

FIG. 3 illustrates an alternative embodiment of a method of theinvention that utilizes hybridization detection of c.

FIG. 4 illustrates another alternative embodiment of a method of theinvention that utilizes hybridization detection of loci.

FIG. 5 illustrates yet another alternative embodiment of a method of theinvention that utilizes hybridization detection of loci.

FIG. 6 illustrates another alternative embodiment of a method of theinvention that utilizes bridging oligonucleotides in combination withfixed sequence oligonucleotides and hybridization detection of c.

FIG. 7 illustrates another alternative embodiment of a method of theinvention that utilizes hybridization detection of loci to detectpolymorphisms.

FIG. 8 illustrates another alternative embodiment of a method of theinvention that utilizes hybridization detection of loci to detectpolymorphisms.

FIG. 9 illustrates another alternative embodiment of a method of theinvention that utilizes hybridization detection of nucleic acid regionsto detect polymorphisms.

FIG. 10 illustrates a method of the invention that utilizeshybridization detection of nucleic acid regions with a bridgingoligonucleotide and dual cleavage.

FIG. 11 of a method of the invention that utilizes hybridizationdetection of nucleic acid regions with a bridging oligonucleotide anddual cleavage to detect polymorphisms.

FIG. 12 illustrates a method of the invention that utilizeshybridization detection of nucleic acid regions resulting from a singlecleavage event and employing differentially labeled universal primers.

FIG. 13 illustrates an alternative method to that illustrated in FIG. 12also utilizing hybridization detection of nucleic acid regions resultingfrom a single cleavage event and employing differentially labeleduniversal primers.

FIG. 14 shows the distribution of assay variability across samples forarrays and next generation sequencing.

DEFINITIONS

The terms used herein are intended to have the plain and ordinarymeaning as understood by those of ordinary skill in the art. Thefollowing definitions are intended to aid the reader in understandingthe present invention, but are not intended to vary or otherwise limitthe meaning of such terms unless specifically indicated.

The term “allele index” refers generally to a series of nucleotides thatcorresponds to a specific SNP. The allele index may contain additionalnucleotides that allow for the detection of deletion, substitution, orinsertion of one or more bases. The allele index may be combined withany other index to create one index that provides information for twoproperties (e.g., sample-identification index, allele-locus index).

“Array” refers to a solid phase support having a surface, preferably butnot exclusively a planar or substantially planar surface, which carriesan array of sites containing nucleic acids such that each site of thearray comprises substantially identical or identical copies ofoligonucleotides or polynucleotides and is spatially defined and notoverlapping with other member sites of the array; that is, the sites arespatially discrete. The array or microarray can also comprise anon-planar interrogatable structure with a surface such as a bead or awell. The oligonucleotides or polynucleotides of the array may becovalently bound to the solid support, or may be non-covalently bound.Conventional microarray technology is reviewed in, e.g., Schena, Ed.,Microarrays: A Practical Approach, IRL Press, Oxford (2000). “Arrayanalysis”, “analysis by array” or “analysis by microarray” refers toanalysis, such as, e.g., sequence analysis, of one or more biologicalmolecules using an array. The term array refers to any format of arrayedsolid substrates, including a microarray, arrayed beads, an array ofmolecules within wells, or “liquid” arrays.

The term “binding pair” means any two molecules that specifically bindto one another using covalent and/or non-covalent binding, and which canbe used, e.g., for attachment of genetic material to a substrate.Examples include, but are not limited to, ligands and their proteinbinding partners, e.g., biotin and avidin, biotin and streptavidin, anantibody and its particular epitope, and the like.

The term “chromosomal abnormality” refers to any genetic variant for allor part of a chromosome. The genetic variants may include but are notlimited to any copy number variant such as duplications or deletions,translocations, inversions, and mutations.

The terms “complementary” or “complementarity” are used in reference tonucleic acid molecules (i.e., a sequence of nucleotides) that arerelated by base-pairing rules.

Complementary nucleotides are, generally, A and T (or A and U), or C andG. Two single stranded RNA or DNA molecules are said to be substantiallycomplementary when the nucleotides of one strand, optimally aligned andwith appropriate nucleotide insertions or deletions, pair with at leastabout 90% to about 95% complementarity, and more preferably from about98% to about 100% complementarity, and even more preferably with 100%complementarity. Alternatively, substantial complementarity exists whenan RNA or DNA strand will hybridize under selective hybridizationconditions to its complement. Selective hybridization conditionsinclude, but are not limited to, stringent hybridization conditions.Stringent hybridization conditions will typically include saltconcentrations of less than about 1 M, more usually less than about 500mM and preferably less than about 200 mM. Hybridization temperatures aregenerally at least about 2° C. to about 6° C. lower than meltingtemperatures (T_(m)).

The term “diagnostic tool” as used herein refers to any composition orsystem of the invention used in combination as, for example, in a systemin order to carry out a diagnostic test or detection system on a patientsample.

The term “hybridization” generally means the reaction by which thepairing of complementary strands of nucleic acid occurs. DNA is usuallydouble-stranded, and when the strands are separated they willre-hybridize under the appropriate conditions. Hybrids can form betweenDNA-DNA, DNA-RNA or RNA-RNA. They can form between a short strand and along strand containing a region complementary to the short one.Imperfect hybrids can also form, but the more imperfect they are, theless stable they will be (and the less likely to form).

As used herein the term “ligase” refers generally to a class of enzymes,DNA ligases (typically T4 DNA ligase), which can link pieces of DNAtogether. The pieces must have compatible ends-either with both of themblunt or with mutually-compatible sticky ends—and the reaction requiresATP. “Ligation” is the process of joining two pieces of DNA together.

The terms “locus” and “loci” as used herein refer to a nucleic acidregion of known location in a genome.

The term “maternal sample” as used herein refers to any sample takenfrom a pregnant mammal which comprises both fetal and maternal cell-freeDNA. Preferably, maternal samples for use in the invention are obtainedthrough relatively non-invasive means, e.g., phlebotomy or otherstandard techniques for extracting peripheral samples from a subject.

The term “oligonucleotides” or “oligos” as used herein refers to linearoligomers of natural or modified nucleic acid monomers, includingdeoxyribonucleotides, ribonucleotides, anomeric forms thereof, peptidenucleic acid monomers (PNAs), locked nucleotide acid monomers (LNA), andthe like, or a combination thereof, capable of specifically binding to asingle-stranded polynucleotide by way of a regular pattern ofmonomer-to-monomer interactions, such as Watson-Crick type of basepairing, base stacking, Hoogsteen or reverse Hoogsteen types of basepairing, or the like. Usually monomers are linked by phosphodiesterbonds or analogs thereof to form oligonucleotides ranging in size from afew monomeric units, e.g., 8-12, to several tens of monomeric units,e.g., 100-200 or more. Suitable nucleic acid molecules may be preparedby the phosphoramidite method described by Beaucage and Carruthers(Tetrahedron Lett., 22:1859-1862 (1981)), or by the triester methodaccording to Matteucci, et al. (J. Am. Chem. Soc., 103:3185 (1981)),both of which are incorporated herein by reference, or by other chemicalmethods such as using a commercial automated oligonucleotidesynthesizer.

As used herein “nucleotide” refers to a base-sugar-phosphatecombination. Nucleotides are monomeric units of a nucleic acid sequence(DNA and RNA). The term nucleotide includes ribonucleoside triphosphatesATP, UTP, CTG, GTP and deoxyribonucleoside triphosphates such as dATP,dCTP, dITP, dUTP, dGTP, dTTP, or derivatives thereof. Such derivativesinclude, for example, [aS]dATP, 7-deaza-dGTP and 7-deaza-dATP, andnucleotide derivatives that confer nuclease resistance on the nucleicacid molecule containing them. The term nucleotide as used herein alsorefers to dideoxyribonucleoside triphosphates (ddNTPs) and theirderivatives. Illustrated examples of dideoxyribonucleoside triphosphatesinclude, but are not limited to, ddATP, ddCTP, ddGTP, ddlTP, and ddTTP.

As used herein the term “polymerase” refers to an enzyme that linksindividual nucleotides together into a long strand, using another strandas a template. There are two general types of polymerase-DNApolymerases, which synthesize DNA, and RNA polymerases, which synthesizeRNA. Within these two classes, there are numerous sub-types ofpolymerases, depending on what type of nucleic acid can function astemplate and what type of nucleic acid is formed.

As used herein “polymerase chain reaction” or “PCR” refers to atechnique for replicating a specific piece of target DNA in vitro, evenin the presence of excess non-specific DNA. Primers are added to thetarget DNA, where the primers initiate the copying of the target DNAusing nucleotides and, typically, Taq polymerase or the like. By cyclingthe temperature, the target DNA is repetitively denatured and copied. Asingle copy of the target DNA, even if mixed in with other, random DNA,can be amplified to obtain billions of replicates. The polymerase chainreaction can be used to detect and measure very small amounts of DNA andto create customized pieces of DNA. In some instances, linearamplification methods may be used as an alternative to PCR.

The term “polymorphism” as used herein refers to any genetic changes orvariants in a loci that may be indicative of that particular loci,including but not limited to single nucleotide polymorphisms (SNPs),methylation differences, short tandem repeats (STRs), and the like.

Generally, a “primer” is an oligonucleotide used to, e.g., prime DNAextension, ligation and/or synthesis, such as in the synthesis step ofthe polymerase chain reaction or in the primer extension techniques. Aprimer may also be used in hybridization techniques as a means toprovide complementarity of a nucleic acid region to a captureoligonucleotide for detection of a specific nucleic acid region.

The term “research tool” as used herein refers to any composition orsystem of the invention used for scientific enquiry, academic orcommercial in nature, including the development of pharmaceutical and/orbiological therapeutics. The research tools of the invention are notintended to be therapeutic or to be subject to regulatory approval;rather, the research tools of the invention are intended to facilitateresearch and aid in such development activities, including anyactivities performed with the intention to produce information tosupport a regulatory submission.

The term “sample” refers to any sample comprising all or a portion ofthe genetic information of an organism, including but not limited tovirus, bacteria, fungus, plants and animals, and in particular mammals.The genetic information that can be interrogated within a genetic sampleincludes genomic DNA (both coding and non-coding regions), mitochondrialDNA, RNA, and nucleic acid products derived from each of these. Suchnucleic acid products include cDNA created from mRNA or products ofpre-amplification to increase the material for analysis.

The term “target genomic region” refers to all or a portion of achromosome or chromosomes, including complete chromosomes,sub-chromosomal regions, groups of loci and single loci.

DETAILED DESCRIPTION OF THE INVENTION

The practice of the techniques described herein may employ, unlessotherwise indicated, conventional techniques and descriptions of organicchemistry, polymer technology, molecular biology (including recombinanttechniques), cell biology, biochemistry, and array technology, which arewithin the skill of those who practice in the art. Such conventionaltechniques include polymer array synthesis, hybridization and ligationof polynucleotides, and detection of hybridization using a label.Specific illustrations of suitable techniques can be had by reference tothe examples herein. However, other equivalent conventional procedurescan, of course, also be used. Such conventional techniques anddescriptions can be found in standard laboratory manuals such as Green,et al., Eds. (1999), Genome Analysis: A Laboratory Manual Series (Vols.I-IV); Weiner, Gabriel, Stephens, Eds. (2007), Genetic Variation: ALaboratory Manual; Dieffenbach, Dveksler, Eds. (2003), PCR Primer: ALaboratory Manual; Bowtell and Sambrook (2003). DNA Microarrays: AMolecular Cloning Manual; Mount (2004), Boinformatics: Sequence andGenome Analysis; Sambrook and Russell (2006), Condensed Protocols fromMolecular Cloning: A Laboratory Manual; and Sambrook and Russell (2002),Molecular Cloning: A Laboratory Manual (all from Cold Spring HarborLaboratory Press); Stryer, L. (1995) Biochemistry (4th Ed.) W.H.Freeman, New York N.Y.; Gait, “Oligonucleotide Synthesis: A PracticalApproach” 1984, IRL Press, London; Nelson and Cox (2000), Lehninger,Principles of Biochemistry 3^(rd) Ed., W. H. Freeman Pub., New York,N.Y.; and Berg et al. (2002) Biochemistry, 5^(th) Ed., W.H. FreemanPub., New York, N.Y., all of which are herein incorporated in theirentirety by reference for all purposes.

Note that as used herein and in the appended claims, the singular forms“a,” “an,” and “the” include plural referents unless the context clearlydictates otherwise. Thus, for example, reference to “an allele” refersto one or more copies of allele with various sequence variations, andreference to “the detection system” includes reference to equivalentsteps and methods known to those skilled in the art, and so forth.

Unless defined otherwise, all technical and scientific terms used hereinhave the same meaning as commonly understood by one of ordinary skill inthe art to which this invention belongs. All publications mentionedherein are incorporated by reference in their entirety for all purposes,including the purpose of describing and disclosing devices, reagents,techniques and methodologies that may be used in or in connection withthe presently described invention.

Where a range of values is provided, it is understood that eachintervening value, between the upper and lower limit of that range andany other stated or intervening value in that stated range isencompassed within the invention. The upper and lower limits of thesesmaller ranges may independently be included in the smaller ranges, andare also encompassed within the invention, subject to any specificallyexcluded limit in the stated range. Where the stated range includes oneor both of the limits, ranges excluding either both of those includedlimits are also included in the invention.

In the following description, numerous specific details are set forth toprovide a more thorough understanding of the present invention. However,it will be apparent to one of skill in the art that the presentinvention may be practiced without one or more of these specificdetails.

In other instances, well-known features and procedures well known tothose skilled in the art have not been described in order to avoidobscuring the invention.

The Invention in General

The invention provides assay methods to identify copy number variants ofnucleic acid regions (including loci, sets of loci and larger targetgenomic regions, e.g., chromosomes), including insertions, deletions,translocations, mutations and polymorphisms in a genetic sample. In oneaspect, the assay methods interrogate loci from two or more targetgenomic regions in a sample using a directed ligation assay followed bydetection of labelled oligonucleotides attached to an array.Quantification of the labelled oligonucleotides allows determination ofan atypical copy number of a particular target genomic region based on acomparison between the quantities of detected loci from the targetgenomic regions (e.g., comparison between two or more portions of asingle chromosome or comparison between two or more differentchromosomes) in the sample or by comparison to a reference chromosomefrom the same or a different sample.

In some embodiments, the method employs directed analysis of targetgenomic regions in a sample using sets of fixed sequenceoligonucleotides that selectively hybridize to loci within two or moretarget genomic regions. The fixed sequence oligonucleotides are directlyor indirectly ligated to create ligation products. The ligation productscorresponding to loci associated with a first target genomic region areassociated with a first detectable label and ligation productscorresponding to loci associated with a second target genomic region areassociated with a second detectable label. If the first and seconddetectable labels are quantified, the relative frequency of each of thefirst and second target genomic regions can be determined. In certainaspects of the invention, the method employs two different labels thatare used to identify two different target genomic regions. In otheraspects of the invention, the method employs three different labelscorresponding to three different target genomic regions, and so on.

The ligation products are detected by hybridization, and in particularby hybridization to an array of capture probes complementary to captureregions present in the ligation products. In certain embodiments, theligation products are detected using “universal arrays” that comprisefeatures having the same or substantially similar capture probes. Incertain other embodiments, the arrays comprises two or more sets ofmultiple features with a common sequence, with each set having adifferent sequence, e.g., an array where up to hundreds of the featureson the array have substantially the same sequence. In either case, thecapture probes on the array are complementary to the capture regions ofthe ligation products rather than to the sequence of the loci or theircomplements. These arrays can be used to interrogate any loci for anytarget genomic region(s) regardless of the sequence of the loci.

The capture regions are preferably introduced to the ligation productsin the fixed sequence oligonucleotides that are used to interrogate theloci in the sample. In some preferred embodiments, the capture regionsare the same amongst all fixed sequence oligonucleotides used, so thatligation products or amplicons or cleavage products thereof from allloci hybridize competitively to capture probes of the same sequence onthe array. In other embodiments, the array is comprised of manydifferent capture probes, and the sets of fixed sequenceoligonucleotides from different loci comprise different capture regions.

FIG. 1 is a flow chart 100 illustrating an exemplary method of theinvention. In step 102, a sample is provided. In step 104, sets of fixedsequence oligonucleotides comprising a label binding region areintroduced to the sample under conditions that allow the fixed sequenceoligonucleotides to hybridize to loci in target genomic regions, and instep 106 the oligonucleotides are hybridized to the target genomicregions. In step 108, the hybridized fixed sequence oligonucleotidesfrom each set are ligated to create ligation products which are thenamplified in step 110 to produce amplicons complementary to the ligationproducts. In step 112, the amplicons are introduced to a hybridizationarray and allowed to hybridize competitively to capture probes on thearray. In step 114, a set of labelled oligonucleotides are introduced tothe amplicons and allowed to hybridize to complementary sequences on theamplicons. Optionally, the labelled oligonucleotides are ligated to thecapture probe on the array (not shown). In step 118, the labels aredetected.

Each fixed sequence oligonucleotide of each set comprises a regioncomplementary to a selected locus (as described in more detail in FIG.2). At least one fixed sequence oligonucleotide of each set furthercomprises a capture region, which may be the same for all sets of fixedsequence oligonucleotides used to interrogate two or more target genomicregions, may be the same for pairs of sets of fixed sequenceoligonucleotides used to interrogate two or more target genomic regions,or may be different between sets of fixed sequence oligonucleotides forindividual target genomic regions. Additionally, depending on theembodiment, one fixed sequence oligonucleotide of a set comprises eithera detectable label or a label binding region for association of thefixed sequence oligonucleotide with the detectable label. In specificembodiments, the label binding region can be a region complementary to alabeled oligonucleotide associated with a detectable label.

In some embodiments, the fixed sequence oligonucleotide of each set thatcomprises the capture region will not comprise the label or labelbinding region; that is, the other fixed sequence oligonucleotide of theset comprises the label or label binding region (see, e.g., exemplaryembodiments illustrated in FIGS. 2, 3 and 5-8); in other embodiments,the fixed sequence oligonucleotide of each set that comprises thecapture region also will comprise the label or label binding region(see, e.g., exemplary embodiment illustrated in FIG. 4).

In certain specific aspects, a first set of fixed sequenceoligonucleotides hybridizes to loci in a first target genomic regionwhile a second set of fixed sequence oligonucleotides hybridizes to lociin a second target genomic region. After ligation to produce ligationproducts, the ligation products are optionally amplified using universalprimers, and then are hybridized to an array. In other embodiments, theamplification product is cleaved (e.g., using a restrictionendonuclease) and a portion of the amplification product comprising thecapture region is introduced to the array for hybridization anddetection. In preferred embodiments, each set of fixed sequenceoligonucleotides used to interrogate a target genomic region containsthe same label or label binding region. That is, all of the fixedsequence oligonucleotides of the first set are associated with a firstlabel, all of the fixed sequence oligonucleotides of the second set areassociated with a second label, and all of the fixed sequenceoligonucleotides in a third set are associated with a third label.

If the fixed sequence oligonucleotides are labeled directly, theligation products, amplicons or cleavage products thereof can behybridized to the capture probes on the array and detected by readoutfrom the labels. If the fixed sequence oligonucleotides instead containa label binding region that is complementary to a labeledoligonucleotide (a “label binding sequence”), a labeled oligonucleotidemust be added to the ligation product or amplicons before detection. Ineither scenario, the labels are then detected and quantified and therelative frequency of each label determined. Quantifying each labelallows for quantification of each target genomic region.

In some embodiments, all sets of first and second fixed sequenceoligonucleotides contain substantially the same capture region. In theseembodiments, because ligation products from all loci from all targetgenomic regions share the same capture region complementary to thecapture probes on the array, the ligation products from each targetgenomic region compete to hybridize to the capture probes on a universalarray.

In other embodiments, the capture probes on an array comprise multiplesequences complementary to different capture regions, and the arraycomprises features that contain these different capture probes. In somesuch embodiments, each capture probe may hybridize to an unique captureregion. In other such embodiments, more than one capture probe,representing loci from different genomic regions, may hybridize to asingle capture region.

In other embodiments, different loci from the same or different targetgenomic regions may be configured to competitively hybridize against oneanother and thus would comprise the same capture region, while otherloci from the same or different target genomic regions may be configuredto competitively hybridize against one another, depending on the assay.

The target genomic regions may be large genomic regions, such as wholechromosomes, or may be smaller genomic regions such as sub-regions of asingle chromosome or sub-regions on different chromosomes, even down toa single locus. Thus, the invention may be used to detect genomicvariations such as aneuploidies and partial aneuploidies, as well asmutations, SNPs, rearrangements, insertions and deletions. In the casewhere whole chromosomes are compared, the first target genomic regionmay be, e.g., chromosome 21, and all loci to be interrogated with thefirst set of fixed sequence oligonucleotides will be from chromosome 21.

In the ligation assay, if the fixed sequence oligonucleotides bind toimmediately adjacent regions in a selected locus, the fixedoligonucleotides may be ligated to create ligation products which areassociated with target genomic region-specific labels. In the case wherefixed sequence oligonucleotides do not bind to immediately adjacentregions within the genomic region—i.e., there is a gap between thehybridized fixed sequence oligonucleotides—the gap can be closed usingprimer extension, and/or one or more bridging oligonucleotides. Once theoligonucleotides are hybridized contiguously, either directly orfollowing an extension operation or introduction of a bridgingoligonucleotide, they may then be ligated to create ligation productswhich are associated with target genomic region-specific labels.

In certain aspects, a first set of fixed sequence oligonucleotides areselective for a first chromosome or first target genomic region and asecond set of fixed sequence oligonucleotides are selective for adifferent chromosome or second target genomic region. FIG. 2 illustratesone embodiment in which each set of labeled fixed sequenceoligonucleotides hybridize to loci on different chromosomes and ligationproducts are evaluated competitively on an array comprising captureprobes. Two sets of labeled fixed sequence oligonucleotides 202, 204 areprovided, each set having a first fixed sequence oligonucleotide 206,208 comprising a sequence that is complementary to a selected locus 210,212 and a label 214, 216 and a second fixed sequence oligonucleotide218, 220 comprising a sequence complementary to the selected locus 222,224 and a capture region 226, 228. The labels 214, 216 are different foreach set of fixed sequence oligonucleotides to allow differentiationbetween the first target genomic region (in this case, a firstchromosome) and the second target genomic region (in this case, a secondchromosome) during detection. In step 230, the sets of fixed sequenceoligonucleotides 202, 204 are introduced to a sample and allowed tohybridize to loci 232, 234 on two different chromosomes. Followinghybridization, the unhybridized fixed sequence oligonucleotidespreferably are separated from the remainder of the sample (not shown).In step 236, the fixed sequence oligonucleotides are ligated to createligation products 238, 240 comprising capture regions 226, 228 andlabels 214, 216. Although the fixed sequence oligonucleotides areillustrated in FIG. 2 as being hybridized adjacently in the loci, theremay also be a gap that can be filled, e.g. using an extension reactionor using a bridging oligonucleotide that hybridizes adjacently betweenthe fixed sequence oligonucleotides. In step 242, the ligation products238, 240 are introduced to a hybridization array 244 comprising aplurality of capture probes 246 wherein the capture regions 226, 228 ofthe ligation products 238, 240 competitively hybridize to the captureprobes 246. In a preferred embodiment, 226 and 228 have substantiallythe same sequence. Following hybridization of the ligation products tothe array, unhybridized ligation products preferably are removed fromthe array (not shown).

The labels 214, 216 can then be detected using an appropriate detectionmechanism depending on the type of label used and the loci correspondingto each of the first and second chromosomes can be quantified todetermine the presence and amount of each chromosome in the geneticsample.

According to the present invention, a “nucleotide” may be unlabeled ordetectably labeled by well-known techniques. Fluorescent labels andtheir attachment to oligonucleotides are described in many reviews,including Haugland, Handbook of Fluorescent Probes and ResearchChemicals, 9th Ed., Molecular Probes, Inc., Eugene Oreg. (2002); Kellerand Manak, DNA Probes, 2nd Ed., Stockton Press, New York (1993);Eckstein, Ed., Oligonucleotides and Analogues: A Practical Approach, IRLPress, Oxford (1991); Wetmur, Critical Reviews in Biochemistry andMolecular Biology, 26:227-259 (1991); and the like. Other methodologiesapplicable to the invention are disclosed in the following sample ofreferences: Fung et al., U.S. Pat. No. 4,757,141; Hobbs, Jr., et al.,U.S. Pat. No. 5,151,507; Cruickshank, U.S. Pat. No. 5,091,519; Menchenet al., U.S. Pat. No. 5,188,934; Begot et al., U.S. Pat. No. 5,366,860;Lee et al., U.S. Pat. No. 5,847,162; Khanna et al., U.S. Pat. No.4,318,846; Lee et al., U.S. Pat. No. 5,800,996; Lee et al., U.S. Pat.No. 5,066,580: Mathies et al., U.S. Pat. No. 5,688,648; and the like.Labeling can also be carried out with quantum dots, as disclosed in thefollowing patents and patent publications: U.S. Pat. Nos. 6,322,901;6,576,291; 6,423,551; 6,251,303; 6,319,426; 6,426,513; 6,444,143;5,990,479; 6,207,392; 2002/0045045; and 2003/0017264. Detectable labelsinclude, for example, radioactive isotopes, fluorescent labels,chemiluminescent labels, bioluminescent labels and enzyme labels.Fluorescent labels of nucleotides may include but are not limitedfluorescein, 5-carboxyfluorescein (FAM),2′7′-dimethoxy-4′5-dichloro-6-carboxyfluorescein (JOE), rhodamine,6-carboxyrhodamine (R6G), N,N,N′,N′-tetramethyl-6-carboxyrhodamine(TAMRA), 6-carboxy-X-rhodamine (ROX), 4-(4′dimethylaminophenylazo)benzoic acid (DABCYL), CASCADE BLUE® (pyrenyloxytrisulfonic acid),OREGON GREEN™ (2′,7′-difluorofluorescein), TEXAS RED™ (sulforhodamine101 acid chloride), Cyanine and5-(2′-aminoethyl)aminonaphthalene-1-sulfonic acid (EDANS). Specificexamples of fluroescently labeled nucleotides include [R6G]dUTP,[TAMRA]dUTP. [R110]dCTP, [R6G]dCTP, [TAMRA]dCTP, [JOE]ddATP, [R6G]ddATP,[FAM]ddCTP, [R110]ddCTP, [TAMRA]ddGTP, [ROX]ddTTP, [dR6G]ddATP,[dR110]ddCTP, [dTAMRA]ddGTP, and [dROX]ddTTP available from PerkinElmer, Foster City, Calif. Fluorolink DeoxyNucleotides, FluoroLinkCy3-dCTP, Fluorolink Cy5-dCTP, FluoroLink FluorX-dCTP, FluoroLinkCy3-dUTP, and FluoroLink Cy5-dUTP available from Amersham, ArlingtonHeights, Ill.; Fluorescein-15-dATP, Fluorescein-12-dUTP,Tetramethyl-rodamine-6-dUTP, IR770-9-dATP, Fluorescein-12-ddUTP,Fluorescein-12-UTP, and Fluorescein-15-2′-dATP available from BoehringerMannheim, Indianapolis, Ind.; and Chromosome. Labeled Nudeotides,BODIPY-FL-14-UTP, BODIPY-FL-4-UTP, BODIPY-TMR-14-UTP,BODIPY-TMR-14-dUTP, BODIPY-T9-14-UTP, BODIPY-TR-14-dUTP, CASCADEBLUES-7-UTP (pyrenyloxytrisulfonic acid-7-UTP), CASCADE BLUE®-7-dUTP(pyrenyloxytrisulfonic acid-7-dUTP), fluorescein-12-UTP,fluorescein-12-dUTP, OREGON GREEN™ 488-5-dUTP(2′,7′-difluorofluorescein-5-dUTP), RHODAMINE GREEN™-5-UTP((5-{2-[4-(aminomethyl)phenyl]-5-(pyridin-4-yl)-1H-i-5-UTP)), RHODAMINEGREEN™-5-dUTP((5-{2-[4-(aminomethyl)phenyl]-5-(pyridin-4-yl)-1H-i-5-dUTP)),tetramethylrhodamine-8-UTP, tetramethylrhodamine-dUTP, TEXAS RED™-5-UTP(sulforhodamine 101 acid chloride-5-UTP), TEXAS RED™-5-dUTP(sulforhodamine 101 acid chloride-5-dUTP), and TEXAS RED™-12-dUTP(sulforhodamine 101 acid chloride-12-dUTP) available from MolecularProbes, Eugene, Oreg.

In certain aspects of the invention, nucleic acids from the sample areassociated with a substrate—e.g., using binding pairs such as, e.g.,biotin and streptavidin, to attach the genetic material to a substratesurface or direct covalent attachment-before adding the sets of fixedsequence oligonucleotides to the sample. Briefly, a first member of abinding pair (e.g., biotin) can be associated with nucleic acids fromthe sample, and the nucleic acids attached to a substrate via a secondmember of the binding pair (e.g., avidin or streptavidin) on the surfaceof the substrate. Attachment of the nucleic acids from the sample can beparticularly useful in moving unhybridized oligonucleotides followinghybridization of the fixed sequence oligonucleotides and/or the bridgingoligonucleotides to the loci. Briefly, the nucleic acids from the samplecan be hybridized to the fixed sequence oligonucleotides, and then thehybridization complexes are subsequently bound to a substrate.Alternatively, the nucleic acids from the sample can be attached to asolid support prior to hybridization of the fixed sequenceoligonucleotides or at the same time. Either way, followinghybridization and attachment of the nucleic acids to a solid support, oralternatively following ligation of the hybridized oligonucleotides, thesurface of the support can be treated to remove any unhybridized orunligated oligonucleotides, e.g., by washing or other removal methodssuch as degradation of oligonucleotides as discussed in Willis et al.,U.S. Pat. Nos. 7,700,323 and 6,858,412. Degradation of theoligonucleotides is a preferred aspect when the two fixed sequenceoligonucleotides are on the same probe such that ligation results in acircularized probe. Exonucleases may then be used to degradenon-circularized nucleic acids, including excess probes and sample DNA.

There are a number of methods that may be used to associate nucleicacids with binding pairs. For example, numerous methods may be used forlabeling nucleic acids with biotin, including random photobiotinylation,end-labeling with biotin, replicating with biotinylated nucleotides, andreplicating with a biotin-labeled primer.

The number of loci analyzed for each chromosome in the methods of theinvention may vary from two to 20,000 or more per target genomic regionanalyzed. In a preferred aspect, the number of loci per target genomicregion is between 48 and 1000. In another aspect, the number of loci pertarget genomic region is at least 100. In another aspect, the number ofloci per target genomic region is at least 400. In another aspect, thenumber of loci per target genomic region is no more than 1000. Inanother aspect, the number of loci per target genomic region is at least500 but no more than 2000.

While the embodiment illustrated in FIG. 2 uses fixed sequenceoligonucleotides coupled directly to a detectable label, a label mayinstead be provided by a separate, labeled oligonucleotide that ishybridized to the ligation products of the fixed sequenceoligonucleotides or amplicons or cleaved amplicon (as described below)thereof, to allow detection. Optionally, the labeled oligonucleotide isligated to the capture probe following hybridization to the fixedsequence oligonucleotides or amplicons or cleaved amplicon. FIG. 3 is anillustration of one embodiment of the invention in which the fixedsequence oligonucleotides are hybridized to loci of interest, ligated,amplified and introduced to an array prior to hybridization of a labeledoligonucleotide and detection of the label. In the method depicted inFIG. 3, two sets of fixed sequence oligonucleotides 302, 304 areprovided, wherein the sets comprise a first fixed sequenceoligonucleotide 306, 308 each comprising sequences complementary to aselected locus 310, 312, label binding region 314, 316 and universalprimer regions 318, 320; and a second fixed sequence oligonucleotide322, 324 comprising sequences complementary to the selected locus 326,328, a capture region 330, 332 and a universal primer region 334, 336.In many embodiments, the capture region 330, 332 comprise substantiallythe same sequence and will both hybridize to the same capture probe onan array. The label binding regions 314, 316 comprise sequences that aredifferent for each set of fixed sequence oligonucleotides 302, 304allowing differential labeling of the fixed sequence oligonucleotidesassociated with the loci for each target genomic region, while thecapture regions 330, 332 are the same for both sets of fixed sequenceoligonucleotides 302, 304 to allow for competitive hybridization of theligation products to capture features on an array. In step 338, the setsof fixed sequence oligonucleotides 302, 304 are introduced to a sampleand allowed to hybridize to loci 340, 342 of different target genomicregions. Following hybridization, unhybridized fixed sequenceoligonucleotides preferably are separated from the remainder of thegenetic sample (not shown).

In step 344, the sets of fixed sequence oligonucleotides 302, 304 areligated to create ligation products 346, 348. In step 358, universalprimers 350, 352, 354 and 356 are introduced to the ligation products346, 348 which bind to the universal primer regions 318, 334, 320, and336, respectively, and create amplicons 360, 362 each comprising captureregions 364, 366 and label binding regions 368, 370. In certainpreferred embodiments, 350 and 354 have substantially the same sequence,which is complementary to both 318 and 320, and 352 and 356 havesubstantially the same sequence, which is complementary to both 334 and336.

The amplicons are introduced to a hybridization array 372 comprising aplurality of capture probes 374 wherein the capture regions 364, 366 ofthe amplicons 360, 362 hybridize to the capture probes 374. In step 378labeled oligonucleotides 380, 382 are introduced to the array 372 wherethe label binding regions 368, 370 of the amplicons 360, 362 hybridizeto target recognition regions 384, 386 of the labeled oligonucleotides380, 382. Optionally, the labelled oligonucleotides are ligated to thecapture probe on the array (not shown). Following hybridization of thelabeled oligonucleotides, unhybridized labeled oligonucleotidespreferably are separated from the array (not shown). The labels can thenbe detected and the loci corresponding to each target genomic regionquantified to provide information on the presence and quantity of eachtarget genomic region in the sample.

In certain embodiments, such as the embodiment shown in FIG. 3, labeledoligonucleotides are hybridized to the fixed sequence oligonucleotides,or amplicons or cleaved amplicons thereof, after the ligation productsor amplicons or cleaved amplicons are hybridized to an array. In othercertain embodiments, the labeled oligonucleotides are hybridized to thefixed sequence oligonucleotides, or amplicons or cleaved ampliconsthereof, prior to hybridization to an array.

To facilitate hybridization of ligation products or amplicons thereof,the size of the ligation products or amplicons may be reduced prior tohybridization to an array. In certain embodiments, the ligation productsare cleaved (e.g., using restriction endonucleases or other enzymaticcleaving mechanisms) to reduce the size of the ligation product to bedetected, leaving, e.g., the capture region and the label binding regionavailable for detection. Detection of a cleaved labeled ligation productor a cleaved amplicon serves as a surrogate in lieu of detecting theentire ligation product.

Reducing the size of the ligation products, amplicons and/or labeledligation products can facilitate binding on the array, e.g., byimproving hybridization kinetics and by decreasing steric hindrance. Incertain embodiments, reduction in the size of the ligation products isaccomplished by cleaving the ligation products or amplicons using arestriction enzyme. For example, in certain embodiments, one of thefixed sequence oligonucleotides in each set of fixed sequenceoligonucleotides comprises a restriction enzyme recognition siteproximal to the capture region or label binding region (or thecorresponding complementary sequences thereof depending on theembodiment). A restriction enzyme can be used to cleave the ligationproduct at the restriction enzyme recognition site leaving the label orlabel binding region and the capture region available for hybridizationand detection. A restriction enzyme recognition site may be located inany position that leaves the capture region and the label binding regionavailable for detection after cleavage. For example, the restrictionenzyme recognition site may be located directly next to the captureregion or the label binding region or within a few bases from either ofthe capture region or label binding region. Preferably cleavage iscarried out prior to hybridization of the labeled oligonucleotide to theligation products or amplicons. In certain other aspects, cleavageoccurs after hybridization to the array for detection.

FIG. 4 is an illustration of a specific embodiment of the invention inwhich the ligation product is cleaved prior to hybridization to anarray. In the method depicted in FIG. 4, two sets of fixed sequenceoligonucleotides 402, 404 are provided. Each set comprises a first fixedsequence oligonucleotide 406, 408 comprising sequences complementary toloci 410, 412, label binding regions 414, 416, capture regions 418, 420,universal primer regions 422, 424 and restriction enzyme recognitionsite regions 426, 428. The label binding regions 414, 416 comprisesequences that are different for the sets of fixed sequenceoligonucleotides 402, 404 to allow differential labeling of fixedsequence oligonucleotides associated with each different target genomicregion, while the capture regions 418, 420 in this embodiment are thesame for both sets of fixed sequence oligonucleotides 402, 404 to allowfor competitive hybridization to the capture features of a hybridizationarray. The restriction enzyme recognition sites 426, 428 can be the samefor both sets of fixed sequence oligonucleotides 402, 404 or differentfor each set depending on the embodiment.

In step 442, the sets of fixed sequence oligonucleotides 402, 404 areintroduced to a sample and allowed to hybridize to elected loci 444,446. Following hybridization and/or ligation, unhybridized fixedsequence oligonucleotides preferably are separated from the remainder ofthe sample (not shown). In step 448, the sets of fixed sequenceoligonucleotides 402, 404 are ligated to create ligation products 450,452. In step 454, universal primers 456, 458, 460 and 462 are introducedto the ligation products 450, 452 which bind to universal primer regions422, 438, 424, and 440, respectively, to create amplicons 464, 466comprising label binding regions 472, 474, capture regions 468, 470 andrestriction enzyme recognition sites 476, 478. In step 480, arestriction enzyme is introduced to the amplicons 464, 466 which bindsto the restriction enzyme recognition site 476, 478 and cleaves theamplicons leaving a cleaved amplicon 482, 484 comprising the labelbinding regions 472, 474 and the capture regions 468, 470. Also at step480, the cleaved amplicons 482, 484 are introduced to a hybridizationarray 486 comprising a plurality of capture probes 488 where the captureregions 488, 470 of the cleaved amplicons 482, 484 competitivelyhybridize to the capture probes 488. In step 492, labeledoligonucleotides 492, 494 are introduced to the array 486 where thelabel binding regions 472, 474 of each cleaved amplicon 482, 484hybridize to target recognition regions 496, 497 of the labeledoligonucleotides 492, 494. Following hybridization of the labeledoligonucleotides to the cleaved amplicons, unhybridized labeledoligonucleotides preferably are separated from the array (not shown).The labels 498, 499 of the labeled oligonucleotides 492, 494 can then bedetected and the cleaved amplicons corresponding to each target genomicregion can be quantified to provide information on the presence andamount of the target genomic regions in the sample. Note that in FIG. 4labeled oligonucleotides 492, 494 abut capture probes 488, and thuslabeled oligonucleotides 492, 494 can be ligated to capture probes 488to, e.g., increase binding stability. Cleaved amplicons 482, 484 canthen be eliminated from the array by washing.

FIG. 5 is an illustration of another specific embodiment of theinvention in which the ligation product is cleaved prior tohybridization to an array. In the method depicted in FIG. 5, two sets offixed sequence oligonucleotides 502, 504 are provided. Each setcomprises a first fixed sequence oligonucleotide 506, 508 comprisingsequences complementary to loci 510, 512, label binding regions 514,516, capture regions 518, 520, universal primer regions 522, 524 andrestriction enzyme recognition site regions 526, 528. The label bindingregions 514, 516 comprise sequences that are different for the sets offixed sequence oligonucleotides 502, 504 to allow differential labelingof fixed sequence oligonucleotides associated with each different targetgenomic region, while the capture regions 518, 520 in this embodimentare the same for both sets of fixed sequence oligonucleotides 502, 504to allow for competitive hybridization to the capture features of ahybridization array. The restriction enzyme recognition sites 526, 528can be the same for both sets of fixed sequence oligonucleotides 502,504 or different for each set depending on the embodiment.

In step 542, the sets of fixed sequence oligonucleotides 502, 504 areintroduced to a sample and allowed to hybridize loci 544, 546. Followinghybridization, unhybridized fixed sequence oligonucleotides preferablyare separated from the remainder of the sample (not shown). In step 548,the sets of fixed sequence oligonucleotides 502, 504 are ligated tocreate ligation products 550, 552. In step 554, universal primers 556,558, 560 and 562 are introduced to the ligation products 550, 552 whichbind to universal primer regions 522, 538, 524, and 540, respectively,to create amplicons 564, 566 comprising label binding regions 572, 574,capture regions 568, 570 and restriction enzyme recognition sites 576,578. In step 580, a restriction enzyme is introduced to the amplicons564, 566 which binds to the restriction enzyme recognition site 576, 578and cleaves the amplicons leaving a cleaved amplicon 582, 584 comprisingthe label binding regions 572, 574 and the capture regions 568, 570. Thecleaved products are bound 580 to their respective labels 582, 584 andintroduced to a hybridization array 588 comprising a plurality ofcapture probes 588 where the capture regions 568, 570 of the cleavedamplicons competitively hybridize 588 to capture probes 590.

In certain aspects of this embodiment of the invention (and asillustrated in and discussed in relation to in FIG. 4), the labeledoligonucleotides may be ligated to the capture probes of thehybridization array to increase the binding stability. This embodimentrequires juxtaposition of the labelled oligonucleotide and the captureprobe. The capture probes, cleaved ligation products and labeledoligonucleotides may be configured so that the labeled oligonucleotidesand capture probes are hybridized adjacently to one another, or thelabeled oligonucleotides and the capture probes can be extended or agap-filling oligonucleotide may be employed as described elsewhereherein.

Detection of Copy Number Variations

As stated above, the invention provides methods to identify copy numbervariants of target genomic regions (relatively short genomic regions,larger genomic regions, sub-chromosomal regions and chromosomes),mutations, and polymorphisms in a sample. The present invention providesparticularly powerful methods for identifying fetal chromosomalaneuploidies in maternal samples comprising both maternal and fetal DNA.

The methods of the invention can also be used to analyze multiple locion multiple chromosomes and average the frequency of the loci from aparticular chromosome together. Normalization or standardization of thefrequencies can be performed for one or more target sequences.

In some aspects, the methods of the invention can be used to sum thefrequencies of the loci on each chromosome for both sources in a mixedsample such as a maternal serum sample, e.g., by detecting an overallsignal of a label, and then comparing the sum of the labels from theloci on one chromosome to the sum of the labels from the loci on anotherchromosome to determine whether a chromosomal abnormality exists.Alternatively, one can analyze subsets of loci on each chromosome todetermine whether a chromosomal abnormality exists. The comparison canbe made either between regions from the same chromosome or betweenregions from different chromosomes.

The data used to determine the frequency of the loci may exclude outlierdata that appear to be due to experimental error, or that have elevatedor depressed levels based on an idiopathic genetic bias within aparticular sample. In one example, the data used for summation mayexclude DNA regions with a particularly elevated frequency in one ormore samples. In another example, the data used for summation mayexclude loci that are found in a particularly low abundance in one ormore samples.

Subsets of loci can be chosen randomly but with sufficient numbers toyield a statistically significant result in determining whether achromosomal abnormality exists. Multiple analyses of different subsetsof loci can be performed within a mixed sample and on different arraysto yield more statistical power. For example, if there are 100 loci forchromosome 21 and 100 loci for chromosome 18, a series of analyses couldbe performed that evaluate fewer than 100 loci for each of thechromosomes on different arrays. In this example, target sequences arenot being selectively excluded.

The quantity of different loci detectable on certain chromosomes mayvary depending upon a number of factors, including generalrepresentation of fetal loci in maternal samples, degradation rates ofthe different loci representing fetal loci in maternal samples, samplepreparation methods, and the like.

Tandem Ligation Assay

In certain aspects, the methods of the invention employ tandem ligationmethods comprising the use of first and second fixed sequenceoligonucleotides that are complimentary to loci in a target genomicregion, e.g., a chromosome of interest or a reference chromosome, andone or more short, bridging oligonucleotides (also called “splint”oligonucleotides or “gap” or “gap-filling” oligonucleotides)complementary to the regions of the loci between and immediatelyadjacent to the first and second fixed sequence oligonucleotides.Hybridization of these oligonucleotides between hybridized fixedsequence oligonucleotides on a selected locus, followed by ligation ofthese oligonucleotides, provides a ligation product which in turnprovides a template for amplification, if desired. A tandem ligationassay tends to be more discriminating than use of a singleoligonucleotide probe or use of only two fixed oligonucleotide probes,as perfect complementarity between the fixed sequence and bridgingoligonucleotides and the selected locus must exist at both ligationsites for ligation to occur.

In preferred aspects, a single bridging oligonucleotide is used whichhybridizes adjacently between the fixed sequence oligonucleotides in thetandem ligation methods. Alternatively in some aspects, the tandemligation methods use sets of two fixed sequence oligonucleotides with aset of two or more bridging oligonucleotides that hybridize adjacentlyin the region of the nucleic acid between the region complementary tothe first and second fixed sequence oligonucleotides. These bridgingoligonucleotides hybridize adjacent to one another and to the fixedsequence oligonucleotides. The two fixed sequence oligonucleotides andtwo bridging oligonucleotides are ligated during the ligation reaction,resulting in a single ligation product which serves as a template foramplification and, ultimately for detection.

In other aspects of the invention, the method employs sets of fixedsequence oligonucleotides that bind to non-adjacent regions within aselected locus, and primer extension is utilized to create a contiguousset of hybridized oligonucleotides prior to the ligation step.Alternatively, the methods may employ sets of fixed sequenceoligonucleotides, one or more bridging oligonucleotides and extension ofone or more of the hybridized oligonucleotides. The combination ofextension and ligation provides a ligation product which in turn servesas a template for amplification, followed by detection andquantification.

In a preferred aspect, the methods of the invention employ a multiplexedreaction with a set of three or more oligonucleotides for each selectedlocus. This general aspect is illustrated in FIG. 6 in which two sets offixed sequence oligonucleotides 602, 604 are provided.

Each set of fixed sequence oligonucleotides 602, 604 comprises firstfixed sequence oligonucleotides 606, 608 each comprising a sequencecomplementary to a nucleic acid region of interest 610, 612, a labelbinding region 614, 616 and a universal primer region 618, 620, andsecond fixed sequence oligonucleotides 622, 624 each comprising asequence complementary to the nucleic acid region of interest 626, 628,a capture region 630, 632 and a universal primer region 634, 636. Instep 638, the sets of fixed sequence oligonucleotides 602, 604 areintroduced to a sample and allowed to specifically hybridize tocomplementary portions of loci 640, 642 in the target genomic regions.Following hybridization, unhybridized fixed sequence oligonucleotidespreferably are separated from the remainder of the genetic sample (notshown). The removal of the unhybridized fixed sequence oligonucleotidesmay alternatively be separated from the remainder of the genetic sampleafter the ligation step below. In step 644, sets of bridgingoligonucleotides 648, 648 are introduced and allowed to specificallyhybridize to the region of the loci between the first fixed sequenceoligonucleotide 606, 608 and second fixed sequence oligonucleotide 622,624. Alternatively, the bridging oligonucleotides 646, 648 can beintroduced simultaneously with the fixed sequence oligonucleotides.

In step 650, the sets of fixed sequence oligonucleotides 602, 604 andbridging oligonucleotides 630, 632 are ligated to create ligationproducts 652, 654 complementary to the loci 640, 642. Also, in step 650,universal primers 658, 660, 662 and 664 are introduced to the ligationproducts 652, 654 where the universal primers bind to the universalprimer regions 618, 634, 620, and 636, respectively, and amplify theligation products to create amplicons 678, 680 comprising label bindingregions 666, 668 and capture regions 662, 664. In certain preferredembodiments, universal primers 658 and 662 have substantially the samesequence, which is complementary to both 618 and 620, and universalprimers 660 and 664 have substantially the same sequence, which iscomplementary to both 634 and 636. The amplicons 678, 680 are introducedto a hybridization array 670 comprising a plurality of capture probes672 wherein the capture regions 662, 664 of the amplicons 658, 660competitively hybridize to target capture regions 674 on the captureprobes 672. In step 676, target recognition regions 682, 684 of thelabeled oligonucleotides 688, 690 specifically hybridize to the labelbinding regions 666, 668 of the amplicons 678, 680. Followinghybridization of the labeled oligonucleotides, unhybridized labeledoligonucleotides preferably are removed from the array (not shown). Thelabels 686, 688 of the labeled oligonucleotides 688, 690 can then bedetected and the loci optionally quantified to provide information onthe presence and amount of each genomic region in the genetic sample.

In certain aspects, the bridging oligonucleotides can be composed of amixture of oligonucleotides with degeneracy in each of the positions, sothat the mixture of bridging oligonucleotides will be compatible withall reactions in the multiplexed assay requiring a bridging of a givenlength. In one example the bridging oligonucleotide is a randomer, whereall combinations of the bridging oligonucleotide are synthesized. As anexample, in the case where a 5-base oligonucleotide is used, the numberof unique bridging oligonucleotide s would be 4{circumflex over( )}5=1024. This would be independent of the number of targeted regionssince all possible bridging oligonucleotide s would be present in thereaction. In another aspect, the bridging oligonucleotides can be ofvarious lengths so that the mixture of oligonucleotides is compatiblewith ligation reactions requiring bridging oligonucleotides of differentlengths.

In yet another aspect, the bridging oligonucleotide can have degeneracyat specific positions—i.e., known polymorphic sites—and the tandemligation reactions are restricted to those that require a specificsequence provided at the polymorphic site.

In another example, the bridging oligonucleotide is specific,synthesized to match the sequences in the gap. As an example, in thecase where a 5-base oligonucleotide is used, the number of uniqueoligonucleotides provided in the assay would be equal to or less thanthe number of loci. A number of bridging oligonucleotides less than thenumber of loci could be achieved if the gap sequence was shared betweentwo or more loci. In one aspect of this example, one might purposefullychoose loci and especially the gap sequences such that there was as muchidentical overlap as possible in the gap sequences, minimizing thenumber of bridging oligonucleotides necessary for the multiplexedreaction.

In another aspect, the sequences of the bridging oligonucleotides aredesigned and the loci are selected so that all loci share the samebase(s) at each end of the bridging oligonucleotide. For instance, onemight choose loci and their gap location such that all of the gapsshared an “A” base at the first position and a “G” base at the lastposition of the gap. Any combination of a first and last base could beutilized, based upon factors such as the genome investigated, thelikelihood of sequence variation in that area, and the like. In aspecific aspect of this example, the bridging oligonucleotides can besynthesized by random degeneracy of bases at the internal positions ofthe bridging oligonucleotide, with nucleotides specific at the first andlast position of the bridging oligonucleotide. In the case of a 5-mer,the second, third and fourth positions would be degenerate, and the twospecific nucleotides at the end of the bridging oligonucleotide would befixed. In this case, the number of unique bridging oligonucleotideswould be 4{circumflex over ( )}3=64.

In the human genome the frequency of the dinucleotide CG is much lowerthan expected by the respective mononucleotide frequencies. Thispresents an opportunity to enhance the specificity of an assay with aparticular mixture of bridging oligonucleotides. In this aspect, thebridging oligonucleotides may be selected to have a 5′ G and a 3′ C.This base selection allows each oligonucleotide to have a high frequencyin the human genome but makes it a rare event for two bridgingoligonucleotides to hybridize adjacent to each other. The probability isthen reduced that multiple oligonucleotides are ligated in locations ofthe genome that are not targeted in the assay.

In certain aspects, the bridging oligonucleotide is added to thereaction after the sets of fixed sequence oligonucleotides have beenhybridized, and following the optional removal of all unhybridized fixedsequence oligonucleotides. The conditions of the hybridization reactionpreferably are optimized near the T_(m) of the bridging oligonucleotideto prevent erroneous hybridization of oligonucleotides that are notfully complementary to the nucleic acid region. If the bridgingoligonucleotides have a T_(m) significantly lower than the fixedsequence oligonucleotides, the bridging oligonucleotide is preferablyadded as a part of the ligase reaction.

The advantage of using short bridging oligonucleotides is that ligationon either end would likely occur only when all bases of the bridgingoligonucleotide match the gap sequence. A further advantage of usingshort bridging oligonucleotides is that the number of different bridgingoligonucleotides necessary could be less than the number of loci,raising the oligonucleotides' effective concentration to allow perfectmatches to happen faster. Fewer numbers of bridging oligonucleotidesprovides the advantages of cost savings and quality control. Theadvantages of using fixed first and last bases and random bases inbetween include the ability to utilize longer bridging oligonucleotidesfor greater specificity while reducing the number of total bridgingoligonucleotides in the reaction.

Detection of Polymorphisms

In certain aspects, the methods of the invention detect one or moretarget genomic regions that comprise a polymorphism. In someembodiments, this methodology is not necessarily designed to identify aparticular allele, e.g., as maternal versus fetal, but rather to ensurethat different alleles corresponding to loci in a target genomic regionare included in the quantification methods of the invention. In certainaspects, however, it may be desirable to both use allelic information tocount all loci in the target genomic region as well as to use theallelic information, e.g., to calculate the amount of fetal DNAcontained within a maternal sample, or identify the percent of alleleswith a particular mutation in a genetic sample from a cancer patient.Thus, the methods of the invention are intended to encompass bothmechanisms for detection of SNP-containing loci for direct determinationof copy number variation through quantification as well as detection ofSNPs for ensuring overall efficiency of the assay.

Thus, in a particular aspect of the invention, allelic discrimination isprovided through the fixed sequence oligonucleotides or through thebridging oligonucleotides used for detection of the loci. In suchembodiments, the label binding region may serve as an allele index thatis embedded in either the first fixed sequence oligonucleotide or thesecond fixed sequence oligonucleotide in a set. In certain specificaspects, an allele index (e.g., label binding region) is present on boththe first and second fixed sequence oligonucleotides to detect two ormore polymorphisms within the loci. The number of fixed sequenceoligonucleotides used in such aspects can correspond to the number ofpossible alleles being assessed for a selected locus, and detection of alabel associated with the allele index can detect presence, amount orabsence of a specific allele in a genetic sample.

FIG. 7 illustrates one aspect of the invention in which polymorphismsare detected using competitive hybridization to an array. In FIG. 7, twosets of fixed sequence oligonucleotides 702, 704 are provided in whichthe first fixed sequence oligonucleotide 706, 708 of each set comprisesa sequence that is complementary to a locus of interest 710, 712comprising for example an A/T or G/C SNP, respectively and a label 714,716. The second fixed sequence oligonucleotide 722, 724 comprises asequence that is complementary to the selected locus 726, 728 and acapture region 730, 732. In some embodiments, fixed sequenceoligonucleotide 706 is the same as fixed sequence oligonucleotide 708except for SNPs 718 and 720 and labels 714 and 716, respectively; andfixed sequence oligonucleotide 722 is the same as fixed sequenceoligonucleotide 724. That is, the difference between the sets of fixedsequence oligonucleotides is the SNP interrogated and the label thatcorresponds to the SNP. In step 734, the sets of fixed sequenceoligonucleotides 702, 704 are introduced to the sample and allowed tospecifically hybridize to locus of interest 736, 738 comprising SNPs740, 742. Following hybridization or ligation, unhybridized fixedsequence oligonucleotides preferably are separated from the remainder ofthe genetic sample (not shown). In step 744, the sets fixed sequenceoligonucleotides 702, 704 are ligated to create ligation products 746,748. It is important to note that in this embodiment the ligation isallele-specific as long as the allele-specific nucleotide is close tothe ligation junction. Typically, the allele-specific nucleotide must bewithin 5 nucleotides of the ligated end; however, in preferred aspects,the allele-specific nucleotide is the terminal base.

In step 750, the ligation products 746, 748 are introduced to ahybridization array 752 comprising a plurality of capture probes 754where the capture regions 730, 732 of the ligation products 746, 748hybridize to the capture probes 754. Following hybridization of theligation products, unhybridized ligation products preferably areseparated from the remainder of the genetic sample (not shown). Thelabels 714, 716 can then be detected and alleles specific to the locicorresponding to each target genomic region are quantified to provideinformation on the presence and amount each allele and target genomicregion in the genetic sample.

In another embodiment, the allele-specific nucleotide is disposed in thefirst and/or second fixed sequence oligonucleotides and bridgingoligonucleotides are used to create ligation products. FIG. 8illustrates this aspect of the invention. In FIG. 8, two sets of fixedsequence oligonucleotides 802, 804 are provided, where both sets offixed sequence oligonucleotides are configured to interrogate differentSNPs at the same selected locus. The first fixed sequenceoligonucleotides 806, 808 of each set comprise a sequence that iscomplementary to a selected locus 810, 812 comprising for example an A/Tor G/C SNP, respectively, a label binding region 814, 816, and auniversal primer region 818, 820; and second fixed sequenceoligonucleotides 826, 828 each comprising sequences that arecomplementary to a selected locus 830, 832, capture region 834, 836 anduniversal primer regions 838, 840. In the polymorphic assays, secondfixed sequence oligonucleotides 826 and 828 have substantially the samesequence and first fixed sequence oligonucleotides 806, 808 havesubstantially the same sequence except for the SNP site and the labelbinding regions 814, 816. In step 842, the sets of fixed sequenceoligonucleotides 802, 804 are introduced to the sample and are allowedto hybridize to the loci 844, 846 comprising SNPs 848, 850. Followinghybridization, unhybridized fixed sequence oligonucleotides preferablyare separated from the remainder of the genetic sample (not shown).

In step 852, a set of bridging oligonucleotides 854, 856 are introducedto the sample and allowed to hybridize to the locus 844, 846 between thefirst and second fixed oligonucleotides of each set. In a preferredembodiment, 854 and 856 have substantially the same sequence. In step858, the hybridized fixed sequence oligonucleotides and hybridizedbridging oligonucleotides are ligated to create ligation products 860,862. In step 864, universal primers 866, 868, 870 and 872 are introducedto the ligation products 860, 862 which bind to universal primer regions818, 838, 820, and 840, respectively and amplify the ligation products860, 862 to produce amplicons 874, 876 comprising label binding regions882, 884 and capture regions 878, 880. The amplicons are introduced to ahybridization array 886 comprising capture probes 888 where the captureregions 878, 880 of the amplicons 874, 876 hybridize to the captureprobes 888. In step 892, labeled oligonucleotides 894, 895 areintroduced to the hybridization array 886 under conditions that allowthe target recognition regions 896, 897 of the labeled oligonucleotides894, 895 to selectively hybridize to the label binding regions 882, 884of the amplicons 874, 876. Following hybridization of the labeledoligonucleotides, unhybridized labeled oligonucleotides preferably areremoved from the array (not shown). The labels 898, 899 can then bedetected and alleles corresponding to the loci corresponding to thetarget genomic regions quantified to provide information on the presenceand amount of the alleles at loci and the target genomic regions in thegenetic sample.

In certain aspects of the invention, allelic discrimination is providedthrough the bridging oligonucleotide. In this aspect, the bridgingoligonucleotide is located over a SNP, preferably located close enoughto one end of a ligation junction so as to provide allelic-specificity.

In one example, both allele bridging oligonucleotide variants arepresent in the same reaction mixture and allele detection results fromsubsequent hybridization of associated labels to hybridization arrays.FIG. 9 illustrates this aspect.

In FIG. 9, two sets of fixed sequence oligonucleotides 902, 904 areprovided where both sets of fixed sequence oligonucleotides areconfigured to interrogate the same selected locus and are the sameexcept for the label binding region. In this embodiment, the bridgingoligonucleotide interrogates the SNP, and the assay must be performed intwo separate vessels at least until after the ligation step has takenplace. The first fixed sequence oligonucleotides 906, 908 in each setcomprise sequences that are complementary to a locus 910, 912, a captureregion 914, 916 and universal primer region 918, 920. The second fixedsequence oligonucleotides 922, 924 of each set comprise sequences thatare complementary to the locus 926, 928, a label binding region 930, 932region and a universal primer region 934, 936. In step 938, the sets offixed sequence oligonucleotides 902, 904 are introduced to the sampleunder conditions that allow each set 902, 904 to specifically hybridizeto the locus 940, 942 wherein the selected locus comprises a SNP 944,946. Following hybridization (or alternatively following ligation) ofthe first and second fixed sequence oligonucleotides, unhybridized fixedsequence oligonucleotides preferably are separated from the remainder ofthe genetic sample (not shown). In step 948, bridging oligonucleotides950, 952 corresponding to an A/T SNP or a G/C SNP are introduced andallowed to bind to the region of the locus 940, 942 between the firstand second fixed sequence oligonucleotides. Alternatively, the bridgingoligonucleotides can be introduced to the sample simultaneously with thefixed sequence oligonucleotides.

In step 954, the hybridized fixed sequence oligonucleotides and bridgingoligonucleotides are ligated to create ligation products 956, 958. Atthis point the two separate assays optionally can be combined into asingle vessel but need not be. In step 960, universal amplificationprimers 962, 964, 966 and 968 are introduced to the ligation products956, 958 where the universal primers bind to universal primer regions918, 934, 920, and 936, respectively, and amplify the ligation products956, 958 to produce amplicons 970, 972, each comprising a label bindingregion 980, 982 and a capture region 974, 976. The amplicons 970, 972are introduced to a hybridization array 984 comprising a plurality ofcapture probes 986 wherein the capture regions 974, 976 of the amplicons970, 972 competitively hybridize to the capture probes 986. In step 990,labeled oligonucleotides 992, 994 are introduced to the array 984 wheretarget recognition regions 996, 997 of the labeled oligonucleotides 992,994 hybridize to label binding regions 980, 982 of the amplicons 970,972. Following hybridization of the labeled oligonucleotides,unhybridized labeled oligonucleotides preferably are removed from thearray (not shown). The labels 998, 999 can then be detected and allelescorresponding to the loci corresponding in turn to the target genomicregions are quantified to provide information on the presence and amountof each allele and target genomic region in the sample.

Additional Embodiments

FIG. 10 is an illustration of another specific embodiment of theinvention in which bridging oligonucleotides are used and the ligationproducts are dually cleaved prior to hybridization to an array. In themethod depicted in FIG. 10, two sets of fixed sequence oligonucleotides1002, 1004 are provided. Each set comprises a first fixed sequenceoligonucleotide 1006, 1008 comprising sequences complementary to loci1010, 1012, label binding regions 1018, 1020, capture regions 1014,1016, universal primer regions 1022, 1024 and restriction enzymerecognition site regions 1026, 1028. The label binding regions 1018,1020 comprise sequences that are different for the sets of fixedsequence oligonucleotides 1002, 1004 to allow differential labeling offixed sequence oligonucleotides associated with each different targetgenomic region, while the capture regions 1014, 1016 in this embodimentare the same for both sets of fixed sequence oligonucleotides 1002, 1004to allow for competitive hybridization to the capture features of ahybridization array. The restriction enzyme recognition sites 1026, 1028can be the same for both cleavage sites on each fixed sequenceoligonucleotide, and/or the same for the different sets of fixedsequence oligonucleotides 1002, 1004 depending on the embodiment. Thereis also a second fixed sequence oligonucleotide 1030, 1032 in each set,where each second fixed sequence oligonucleotide comprises a sequencecomplementary to the loci 1034, 1036 and a universal primer region 1038,1040.

In step 1042, the sets of fixed sequence oligonucleotides 1002, 1004 areintroduced to a sample and allowed to hybridize loci 1044, 1046.Following hybridization (or alternatively ligation), unhybridized fixedsequence oligonucleotides preferably are separated from the remainder ofthe sample (not shown). In step 1048, a bridging oligonucleotide 1026,1028 is added to each set and hybridized adjacently between thehybridized fixed sequence oligonucleotides. At step 1054, the sets ofoligonucleotides are ligated to create ligation products 1050, 1052. Instep 1054, universal primers 1056, 1058, 1060 and 1062 are introduced tothe ligation products 1050, 1052 which bind to universal primer regions1022, 1038, 1024, and 1040, respectively, to create amplicons 1064, 1066comprising label binding regions 1018, 1020, capture regions 1014, 1016,and restriction enzyme recognition sites 1072, 1074, 1076, and 1078. Instep 1070, one or more restriction enzymes are introduced to theamplicons 1064, 1066 and the amplicons are dually cleaved to create acleaved amplicon comprising the label binding regions 1018, 1020 and thecapture regions 1014, 1016. The cleaved products are introduced to ahybridization array 1086 comprising a plurality of capture probes 1088where the capture regions 1014, 1016 of the cleaved ampliconscompetitively hybridize to capture probes 1090. The hybridized captureregions are then introduced 1088 to the labeled oligonucleotides 1082,1084, which bind to complementary sequences on the cleaved products andare detected.

FIG. 11 is an illustration of another specific embodiment of theinvention in which bridging oligonucleotides are used to identifydifferent alleles in the same locus and the ligation products are duallycleaved prior to hybridization to an array. In the method depicted inFIG. 11, two sets of fixed sequence oligonucleotides 1102, 1104 areprovided for a single locus having a possible polymorphism. Each setcomprises a first fixed sequence oligonucleotide 1106, 1108 comprisingsequences complementary to one allele of the selected locus 1110, 1112,label binding regions 1118, 1120, capture regions 1114, 1116, universalprimer regions 1122, 1124 and restriction enzyme recognition siteregions 1126, 1128. The label binding regions 1118, 1120 comprisesequences that are different for the sets of fixed sequenceoligonucleotides 1102, 1104 to allow differential labeling of fixedsequence oligonucleotides associated with each different allele, whilethe capture regions 1114, 1116 in this embodiment are the same for bothsets of fixed sequence oligonucleotides 1102, 1104 to allow forcompetitive hybridization to the capture features of a hybridizationarray. The restriction enzyme recognition sites 1126, 1128 can be thesame for both cleavage sites on each fixed sequence oligonucleotide,and/or the same for the different sets of fixed sequenceoligonucleotides 1102, 1104 depending on the embodiment. There is also asecond fixed sequence oligonucleotide 1130, 1132 in each set, where eachsecond fixed sequence oligonucleotide comprises a sequence complementaryto the selected locus 1134, 1138 and a universal primer region 1138,1140.

In step 1142, the sets of fixed sequence oligonucleotides 1102, 1104 areintroduced to a sample and allowed to hybridize to the selected locus1144, 1146. Following hybridization (or alternatively ligation),unhybridized fixed sequence oligonucleotides preferably are separatedfrom the remainder of the sample (not shown). In step 1148, a bridgingoligonucleotide 1126, 1128 is added to each set and hybridizedadjacently between the hybridized fixed sequence oligonucleotides. Atstep 1154, the sets of oligonucleotides are ligated to create ligationproducts 1150, 1152. In step 1154, universal primers 1156, 1158, 1160and 1162 are introduced to the ligation products 1150, 1152 which bindto universal primer regions 1122, 1138, 1124, and 1140, respectively, tocreate amplicons 1164, 1166 comprising label binding regions 1118, 1120,capture regions 1114, 1116, and restriction enzyme recognition sites1172, 1174, 1176, and 1178. In step 1170, one or more restrictionenzymes are introduced to the amplicons 1164, 1166 and the ampliconsdually cleaved to create a cleaved amplicon comprising the label bindingregions 1118, 1120 and the capture regions 1114, 1116. The cleavedproducts are introduced to a hybridization array 1186 comprising aplurality of capture probes 1188 where the capture regions 1118, 1120 ofthe cleaved amplicons competitively hybridize to capture probes 1190.The hybridized capture regions are then introduced 1188 to the labeledoligonucleotides 1182, 1184, which bind to complementary sequences onthe cleaved products and are detected to differentiate between thedifferent alleles of the locus.

FIG. 12 is an illustration of another specific embodiment of theinvention in which bridging oligonucleotides are used and the ligationproducts are cleaved prior to hybridization to an array. In thisembodiment, the primers used to amplify the ligation products aredifferentially labeled. In the method depicted in FIG. 12, two sets offixed sequence oligonucleotides 1202, 1204 are provided. Each setcomprises a first fixed sequence oligonucleotide 1206, 1208 comprisingsequences complementary to loci 1210, 1212, primer binding regions 1218,1224 (which are different), capture regions 1214, 1220, and restrictionenzyme recognition site regions 1226, 1228. The primer binding regions1218, 1224 comprise sequences that are different for the sets of fixedsequence oligonucleotides 1202, 1204 to allow differential labeling offixed sequence oligonucleotides associated with each different targetgenomic region, while the capture regions 1214, 1220 in this embodimentare the same for both sets of fixed sequence oligonucleotides 1202, 1204to allow for competitive hybridization to the capture features of ahybridization array. The restriction enzyme recognition sites 1226, 1228can be the same for both cleavage sites on each fixed sequenceoligonucleotide, and/or the same for the different sets of fixedsequence oligonucleotides 1302, 1304 depending on the embodiment. Thereis also a second fixed sequence oligonucleotide 1230, 1232 in each set,where each second fixed sequence oligonucleotide comprises a sequencecomplementary to the loci 1234, 1236 and a primer region 1238, 1240.

In step 1242, the sets of fixed sequence oligonucleotides 1202, 1204 areintroduced to a sample and allowed to hybridize loci 1244, 1246.Following hybridization (or alternatively ligation), unhybridized fixedsequence oligonucleotides preferably are separated from the remainder ofthe sample (not shown). In step 1248, a bridging oligonucleotide 1226,1228 is added to each set and hybridized adjacently between thehybridized fixed sequence oligonucleotides. At step 1254, the sets ofoligonucleotides are ligated to create ligation products 1250, 1252. Instep 1254, primers 1256, 1258, 1260 and 1262 are introduced to theligation products 1250, 1252 which bind to primer regions 1218, 1238,1224, and 1240, respectively, to create amplicons 1264, 1266 comprisingprimer binding regions 1278, 1284, capture regions 1276, 1280, andrestriction enzyme recognition sites 1272 and 1274. Primers 1256 and1260 are differentially labeled thus allowing for differentiationbetween amplicons from the different loci once the amplicons arehybridized to an array. In step 1270, one or more restriction enzymesare introduced to the amplicons 1264, 1266 and the amplicons are cleavedto create a cleaved amplicon comprising the primer binding regions 1278,1284 and the capture regions 1276, 1280. The cleaved products areintroduced to a hybridization array 1286 at step 1290 comprising aplurality of capture probes 1288 where the capture regions 1276, 1280 ofthe cleaved amplicons competitively hybridize to capture probes 1288.

FIG. 13 is an illustration of another specific embodiment of theinvention in which bridging oligonucleotides are used and the ligationproducts are cleaved prior to hybridization to an array. FIG. 13illustrates an embodiment very similar to that in FIG. 12. In thisembodiment, the primers used to amplify the ligation products aredifferentially labeled. In the method depicted in FIG. 13, two sets offixed sequence oligonucleotides 1302, 1304 are provided. Each setcomprises a first fixed sequence oligonucleotide 1306, 1308 comprisingsequences complementary to loci 1310, 1312, primer binding regions 1318,1324 (which are different), capture regions 1314, 1320 (which are thesame), and restriction enzyme recognition site regions 1326, 1328. Theprimer binding regions 1318, 1324 comprise sequences that are differentfor the sets of fixed sequence oligonucleotides 1302, 1304 to allowdifferential labeling of fixed sequence oligonucleotides associated witheach different target genomic region, while the capture regions 1314,1320 in this embodiment are the same for both sets of fixed sequenceoligonucleotides 1302, 1304 to allow for competitive hybridization tothe capture features of a hybridization array. The restriction enzymerecognition sites 1326, 1328 can be the same for both cleavage sites oneach fixed sequence oligonucleotide, and/or the same for the differentsets of fixed sequence oligonucleotides 1302, 1304 depending on theembodiment. There is also a second fixed sequence oligonucleotide 1330,1332 in each set, where each second fixed sequence oligonucleotidecomprises a sequence complementary to the loci 1334, 1326 and a primerregion 1338, 1340.

In step 1342, the sets of fixed sequence oligonucleotides 1302, 1304 areintroduced to a sample and allowed to hybridize loci 1344, 1346.Following hybridization (or alternatively ligation), unhybridized fixedsequence oligonucleotides preferably are separated from the remainder ofthe sample (not shown). In step 1348, a bridging oligonucleotide 1326,1328 is added to each set and hybridized adjacently between thehybridized fixed sequence oligonucleotides. At step 1354, the sets ofoligonucleotides are ligated to create ligation products 1350, 1352. Instep 1354, primers 1356, 1358, 1360 and 1362 are introduced to theligation products 1350, 1352 which bind to primer regions 1318, 1338,1324, and 1340, respectively, to create amplicons 1364, 1366 comprisingprimer binding regions 1378, 1384, capture regions 1376, 1380, andrestriction enzyme recognition sites 1372 and 1374. Primers 1356 and1360 are differentially labeled thus allowing for differentiationbetween amplicons from the different loci once the amplicons arehybridized to an array. In step 1370, one or more restriction enzymesare introduced to the amplicons 1364, 1366 and the amplicons are cleavedto create a cleaved amplicon comprising the primer binding regions 1378,1384 and the capture regions 1376, 1380. The cleaved products areintroduced to a hybridization array 1386 at step 1390 comprising aplurality of capture probes 1388 where the capture regions 1376, 1380 ofthe cleaved amplicons competitively hybridize to capture probes 1388.

In the embodiments of FIGS. 10-13, the cleavage amplicons introduced tothe array do not include any portion of the target genomic region or asequence complementary thereof. The capture regions are used inassociation with different target genomic regions, and the labeledoligonucleotide is used to differentiate between regions. In thismanner, the cleaved amplicons competitively hybridize to common captureprobes, and the quantitation of the target genomic regions is determinedby the detected labels corresponding to cleaved products that bind tothe array.

As described previously, the embodiments of the present invention shownin FIGS. 2-13 depict two separate fixed sequence oligonucleotides usedto interrogate each locus or allele. However, in some aspects, however,a single probe can be used which comprises two or more distinctnon-adjacent fixed sequence oligonucleotides that are complementary tothe loci including precircle probes. Such precircle probes aredescribed, e.g., by Uzardi in U.S. Pat. Nos. 5,854,033 and 6,316,229,and can be linearized prior to hybridization to the array by, e.g.,including a site for a restriction endonuclease in the probe.

Universal Amplification

In certain aspects of the invention, universal amplification is used toamplify the ligation products following hybridization and ligation ofthe fixed sequence oligonucleotides, either directly or followingextension or the introduction of a bridging oligonucleotide. In amultiplexed assay system, amplification preferably is done throughuniversal amplification using universal primers that hybridize touniversal primer regions on the first and second fixed sequenceoligonucleotide of each set. The universal primer regions from the fixedsequence oligonucleotides become a part of the ligation products, wherethe ligation products can then be amplified in a single universalamplification reaction. Although these universal primer sequencespreferably are introduced via the fixed sequence oligonucleotides, theymay also be added to the proximal ends of the ligation products vialigation. The introduction of universal primer regions to the fixedsequence oligonucleotides allows a subsequent controlled universalamplification of all or a portion of the ligation products prior toarray hybridization. The amplicon produced from this amplificationprocess can be used directly or further processed prior to introductionto the array for detection. In specific embodiments, the amplicon iscleaved, and the portion comprising the capture region is introduced tothe array for hybridization and detection. Bias and variability can beintroduced during DNA amplification, such as that seen during polymerasechain reaction (PCR). In cases where an amplification reaction ismultiplexed, there is the potential that loci will amplify at differentrates or efficiency. Sets of primers for a given locus may behavedifferently based on sequence context of the primer and template DNA,buffer conditions, and other conditions. In certain aspects, theuniversal primer regions used in the methods are designed to becompatible with conventional multiplexed assay methods that utilizegeneral priming mechanisms to analyze large numbers of nucleic acidssimultaneously. Such “universal” priming methods allow for efficient,high volume analysis of the quantity of nucleic acid regions present ina genetic sample, and allow for comprehensive quantification of thepresence of nucleic acid regions within such a sample.

The entirety of a ligation reaction or an aliquot of the ligationreaction may be used for universal amplification. Using an aliquotallows parallel amplification reactions to be undertaken using the sameor different conditions (e.g., polymerase, buffers, and the like), e.g.,to ensure that bias is not inadvertently introduced due to experimentalconditions. In addition, variations in primer concentrations may be usedto effectively limit the number of sequence specific amplificationcycles. Examples of multiplexing assay methods include, but are notlimited to, those described in Oliphant et al., U.S. Pat. No. 7,582,420.

As described in detail herein, many methods of the invention utilizecoupled reactions for multiplex detection where oligonucleotides from anearly phase of the multi-step process contain sequences that may be usedin a later phase of the process. Processes known in the art foramplifying and/or detecting nucleic acids in samples can be used, aloneor in combination, including but not limited to the methods describedbelow. In certain aspects, the methods of the invention utilize one ofthe following combined selective and universal amplification techniques:(1) LDR coupled to PCR; (2) primary PCR coupled to secondary PCR coupledto LDR; and (3) primary PCR coupled to secondary PCR. Each of thesecombinations of techniques can utilize probe regions from an early phasein the process that may be used as a primer sequence in a later phase ofthe process.

Barany et al., U.S. Pat. Nos. 6,852,487, 6,797,470, 6,576,453,6,534,293, 6,506,594, 6,312,892, 6,268,148, 6,054,564, 6,027,889,5,830,711, 5,494,810, describe the use of the ligase chain reaction(LCR) assay for the detection of specific sequences of nucleotides in avariety of nucleic acid samples.

Barany et al., U.S. Pat. Nos. 7,807,431, 7,455,965, 7,429,453,7,364,858, 7,358,048, 7,332,285, 7,320,865, 7,312,039, 7,244,831,7,198,894, 7,166,434, 7,097,980, 7,083,917, 7,014,994, 6,949,370,6,852,487, 6,797,470, 6,576,453, 6,534,293, 6,506,594, 6,312,892, and6,268,148 describe the use of the ligase detection reaction withdetection reaction (“LDR”) coupled with polymerase chain reaction(“PCR”) for nucleic acid detection.

Barany et al., U.S. Pat. Nos. 7,556,924 and 6,858,412, describe the useof padlock probes (also called “precircle probes” or “multi-inversionprobes”) with coupled ligase detection reaction (“LDR”) and polymerasechain reaction (“PCR”) for nucleic acid detection.

Barany et al., U.S. Pat. Nos. 7,807,431, 7,709,201, and 7,198, 814describe the use of combined endonuclease cleavage and ligationreactions for the detection of nucleic acid sequences.

Willis et al., U.S. Pat. Nos. 7,700,323 and 6,858,412, describe the useof precircle probes in multiplexed nucleic acid amplification, detectionand genotyping.

Ronaghi et al., U.S. Pat. No. 7,622,281 describes amplificationtechniques for labeling and amplifying a nucleic acid using an adaptercomprising a unique primer and a barcode.

The nucleic acid regions of interest are identified using hybridizationtechniques and arrays. Methods for conducting polynucleotidehybridization assays for detection are well-developed and well-known inthe art. Hybridization assay procedures and conditions will varydepending on the application and are selected in accordance with thegeneral binding methods known including those referred to in: Maniatiset al. Molecular Cloning: A Laboratory Manual (2^(nd) Ed. Cold SpringHarbor, N.Y., 1989); Berger and Kimmel Methods in Enzymology, Vol. 152,Guide to Molecular Cloning Techniques (Academic Press, Inc., San Diego,Calif., 1987); Young and Davis, P.N.A.S, 80: 1194 (1983). Methods andapparatus for carrying out repeated and controlled hybridizationreactions have been described in U.S. Pat. Nos. 5,871.928, 5,874,219,6,045,996 and 6,386,749, 6,391,623 each of which are incorporated hereinby reference.

In some embodiments, the arrays comprise multiple substrates insolution, such as those taught, e.g., in U.S. Appln No. 20140057269 andU.S. Appln No. 20140042366 and U.S. Appln No. 20140024550.

The present invention also contemplates signal detection ofhybridization between ligands in certain preferred aspects. See U.S.Pat. Nos. 5,143,854, 5,578,832; 5,631,734; 5,834,758; 5,936,324;5,981,956; 6,025,601; 6,141,096; 6,185,030; 6,201,639; 6,218,803; and6,225,625, in U.S. Patent application 60/364,731 and in PCT ApplicationPCT/US99/06097 (published as WO99/47964).

Methods and apparatus for signal detection and processing of intensitydata are disclosed in, for example, U.S. Pat. Nos. 5,143,854, 5,547,839,5,578,832, 5,631,734, 5,800,992, 5,834,758; 5,856,092, 5,902,723,5,936,324, 5,981,956, 6,025,601, 6,090,555, 6,141,096, 6,185,030,6,201,639; 6,218,803; and 6,225,625, in U.S. Patent application60/364,731 and in PCT Application PCT/US99/06097 (published asWO99/47964).

In certain aspects, the capture region of the ligation products from asingle sample or the amplicons thereof contain index sequences thatidentify the ligation products as being from a particular sample. Thefeatures of the array will comprise capture probes that includesequences complementary to the index sequences of the different samplesto allow identification of the loci from a particular sample from whichthe loci originated.

Estimation of Fetal DNA Proportion in a Maternal Sample

The proportion of fetal DNA in a maternal sample may be used as a partof the risk calculation of the present invention, as fetal proportionprovides important information on the expected statistical presence ofchromosomal dosage. Variation from the expected statistical presence maybe indicative of fetal aneuploidy, an in particular a fetal trisomy ormonosomy of a particular chromosome.

Any methods known in the art to estimate the percentage of fetal DNA ina maternal sample may be used, including quantifying Y sequences if thefetus is male or looking at epigenetic markers (see, e.g., Chim, et al.,PNAS USA, 102:14753-58 (2005)). Using fetal proportion as one componentof the risk calculation is particularly helpful in circumstances wherethe level of fetal DNA in a maternal sample is low. Further, knowledgeof the fetal DNA percentage may be used to determine what if anyadditional analyses can be performed on the sample, as it may be thecase at a certain lower bound of fetal DNA percentage a system is notable to reliably perform analysis. In other aspects, determining thefetal DNA proportion in a maternal sample may additionally affect thelevel of certainty or power in detecting a fetal aneuploidy.

Although the following methods are described for determination of atotal proportion of fetal content in a maternal sample, the proportioncan also be determined on a chromosome by chromosome basis. For instancefrequency information for fetal chromosome 21 can be determined ascompared to fetal chromosome 18. In another example, two or morechromosomes can be used in detecting a fetal proportion, e.g., frequencyof loci on chromosomes 1 and 2 can be used. In certain aspects, thechromosome used for determining fetal proportion is the chromosomeinterrogated for possible aneuploidy. In another aspect, thechromosome(s) used for determining fetal proportion are specifically notthe chromosome interrogated for possible aneuploidy.

The DNA from a fetus will have approximately 50% of its loci inheritedfrom the mother and approximately 50% its loci inherited from thefather. Determining which genetic loci are contributed to the fetus fromnon-maternal sources (informative loci) allows the estimation of fetalDNA proportion in a maternal sample, and thus provides information usedto calculate statistically significant differences in chromosomaldosages for chromosomes of interest.

In certain aspects, determination of non-maternal polymorphisms isachieved through targeted SNP and/or mutation analysis to estimate thepercentage of fetal DNA in a maternal sample—a process which isparticularly adaptable to the array analysis of the present invention.The percent fetal cell free DNA in a maternal sample can be quantifiedusing multiplexed SNP detection without prior knowledge of the maternalor paternal genotype. In this aspect, two or more selected polymorphicnucleic acid regions with a known SNP in each region are used. In apreferred aspect, the selected polymorphic nucleic acid regions arelocated on an autosomal chromosome that is unlikely to be aneuploid,e.g., not chromosomes 21, 18, or 13. The selected polymorphic nucleicacid regions from the maternal sample (e.g., plasma) are amplified. In apreferred aspect, the amplification is universal; and in a preferredembodiment, the selected polymorphic nucleic acid regions are amplifiedin one reaction in one vessel, and in a preferred embodiment, theselected polymorphic nucleic acid regions are amplified in one reactionin one vessel with the ligation products of the fixed sequenceoligonucleotides used to determine a chromosomal aneuploidy. Each alleleof the selected polymorphic nucleic acid regions in the maternal sampleis determined and quantified. In a preferred aspect, high throughputsequencing is used for such determination and quantification.

Loci are thus identified where the maternal and non-maternal genotypesare different; e.g., the maternal genotype is homozygous and thenon-maternal genotype is heterozygous. This identification ofinformative loci is accomplished by observing a high frequency of oneallele (>80%) and a low frequency (<20% and >0.15%) of the other allelefor a particular selected nucleic acid region. The use of multiple lociis particularly advantageous as it reduces the amount of variation inthe measurement of the abundance of the alleles between loci. All or asubset of the loci that meet this requirement are used to determinefetal contribution through statistical analysis. In one aspect, fetalcontribution is determined by summing the low frequency alleles from twoor more loci together, dividing by the sum of the low and high frequencyalleles and multiplying by two.

For many alleles, maternal and non-maternal sequences may be homozygousand identical, and as this information therefore does not distinguishbetween maternal and non-maternal DNA it is not useful in thedetermination of percent fetal DNA in a maternal sample. The presentinvention utilizes allelic information where there is a distinguishabledifference between the non-maternal and maternal DNA (e.g., a fetalallele containing at least one allele that differs from the maternalallele) in calculations of percent fetal DNA. Data pertaining to allelicregions that are the same for maternal and non-maternal DNA are thus notselected for analysis, or are removed from the pertinent data prior toestimation of the fetal DNA proportion so as not to mask the usefuldata. Additional exemplary processes for quantifying fetal DNA inmaternal plasma can be found, e.g., in Chu, et al., Prenat. Diagn.,30:1228-29 (2010), which is incorporated herein by reference.

Data Analysis

Once percent fetal cell free DNA has been calculated, this data may becombined with methods for aneuploidy detection to determine thelikelihood that a fetus may contain an aneuploidy. In one aspect, ananeuploidy detection method that utilizes analysis of random DNAsegments is used, such as that described in, e.g., Quake, U.S. Ser. No.11/701,686; and Shoemaker et al., U.S. Ser. No. 12/230,628. In apreferred aspect, aneuploidy detection methods that utilize analysis ofselected nucleic acid regions are used. In this aspect, the percentfetal cell free DNA for a sample is calculated. The chromosomal ratiofor that sample, a chromosomal ratio for the normal population and avariation for the chromosomal ratio for the normal population isdetermined, as described herein. Alternatively, the calculatedchromosomal ration uses an expectation for a chromosomally normal sampleand an expectation for an aneuploid sample. The calculated chromosomalratio for a sample is then compared to those expectations.

In one preferred aspect, the chromosomal ratio and its variation for thenormal population are determined from normal samples that have a similarpercentage of fetal DNA. An expected aneuploid chromosomal ratio for aDNA sample with that percent fetal cell free DNA is calculated by addingthe percent contribution from the aneuploid chromosome. The chromosomalratio for the sample may then be compared to the chromosomal ratio forthe normal population and to the expected aneuploid chromosomal ratio todetermine statistically, using the variation of the chromosomal ratio,if the sample is more likely normal or aneuploid, and the statisticalprobability that it is one or the other.

In a preferred aspect, the selected regions of a maternal sample includeboth regions for estimation of fetal DNA content as well asnon-polymorphic regions from two or more chromosomes to detect a fetalaneuploidy in a single reaction. The single reaction helps to minimizethe risk of contamination or bias that may be introduced during varioussteps in the assay system which may otherwise skew results whenutilizing fetal DNA content to help determine the presence or absence ofa chromosomal abnormality.

In other aspects, a selected nucleic acid region or regions may beutilized both for estimation of fetal DNA content as well as detectionof fetal chromosomal abnormalities. The alleles for selected nucleicacid regions can be used to estimate fetal DNA content and these sameselected nucleic acid regions can then be used to detect fetalchromosomal abnormalities ignoring the allelic information. Utilizingthe same selected nucleic acid regions for both fetal DNA content anddetection of chromosomal abnormalities may further help minimize anybias due to experimental error or contamination.

In one embodiment, fetal source contribution in a maternal sampleregardless of fetal gender is measured using autosomal SNPs (see,Sparks, et al., Am. J. Obstet & Gyn., 206:319.e1-9 (2012)). Theprocesses utilized do not require prior knowledge of paternal genotype,as the non-maternal alleles are identified during the methods withoutregard to knowledge of paternal inheritance. A maximum likelihoodestimate using the binomial distribution may be used to calculate theestimated fetal nucleic acid contribution across several informativeloci in each maternal sample. The processes for calculation of fetalacid contribution used are described, for example, in US Pub. No.2013/0024127. The polymorphic regions used for determination of fetalcontribution may be from chromosomes 1-12, and preferably do not targetthe blood group antigens. The estimate of fetal contribution from thepolymorphic assays is used to define expected response magnitudes when atest chromosome is trisomic, which informs the statistical testing. Thetest statistic may consist of two components: a measure of deviationfrom the expected proportion when the sample is disomic; and a measureof deviation from the expected proportion when the sample is trisomic.Each component is in the form of a Wald statistic (e.g., Harrell,Regression modeling strategies, (2001, Springer-Verlag), Sections 9.2.2and 10.5) which compares an observed proportion to an expectedproportion and divides by the variation of the observation.

The statistic Wj may be used to measure the deviation from expectationwhen the sample j is disomic, and is defined as

${W_{j} = \frac{p_{j} - p_{0}}{\sigma_{pj}}},$

where pj and p0 are defined as described supra with the Z statistic, andσ_(Pj) is the standard deviation of the observed proportion ofrepresentation for a given chromosome of interest. The standarddeviation may be estimated using parametric bootstrap sampling to createa distribution of pj proportions based on the mean counts and standarderrors for our chromosomes of interest. The second statistic is Ŵ_(j),which replaces p0 with the fetal fraction adjusted reference proportion{circumflex over (p)}_(j) is defined as

${{\hat{p}}_{j} = \frac{\left( {1 + {0.5f_{j}}} \right)p_{0}}{\left( {\left( {1 + {0.5f_{j}}} \right)p_{0}} \right)\left( {1 - p_{0}} \right)}},$

where f_(i) is the fetal fraction for sample j and p₀ is the referenceproportion as before. This adjustment accounts for the increasedrepresentation of a test chromosome when the fetus was trisomic. Becausethis variance of counts across many loci is measured as a natural resultof using multiple non-polymorphic assays for there test chromosomes, allestimates are taken within a nascent data set and do not requireexternal reference samples or historical information with normalizingadjustments to control for process drift as is typically required forvariance around the expected proportion.

The final statistic used was S_(j)=W_(j)+Ŵ_(j). Conceptually, deviationsfrom disomic expectation and trisomic expectation are simultaneouslyevaluated and summarized into this single statistic. The particularadvantage of combining these two indicators is that while deviation fromdisomy might be high, it may not reach the deviation expected fortrisomy at a particular fetal contribution level. The Ŵ_(j) componentwill be negative in this case, in effect penalizing the deviation fromdisomy. An S_(j)=0 indicated an equal chance of being disomic vs.trisomic.

Computer Implementation of the Processes of the Invention

According to an exemplary embodiment, a computer executes a softwarecomponent that calculates fetal proportion and applies this informationto the values of the dosage of genomic regions and/or chromosomes. Inone embodiment, the computer may comprise a personal computer, but thecomputer may comprise any type of machine that includes at least oneprocessor and memory.

The output of the software component comprises a report with a value ofprobability that a genomic region and/or a chromosome has a dosageabnormality. In a preferred aspect this report is an odds ratio of avalue of the likelihood that a region or chromosome has two copies(e.g., is disomic) and a value of the likelihood that a region orchromosome has more copies (e.g., is trisomic) or less copies (e.g., ismonosomic) copies. The report may be paper that is printed out, orelectronic, which may be displayed on a monitor and/or communicatedelectronically to users via e-mail, FTP, text messaging, posted on aserver, and the like.

Although the normalization process of the invention is shown as beingimplemented as software, it can also be implemented as a combination ofhardware and software. In addition, the software for normalization maybe implemented as multiple components operating on the same or differentcomputers.

Both the server and the computer may include hardware components oftypical computing devices, including a processor, input devices (e.g.,keyboard, pointing device, microphone for voice commands, buttons,touchscreen, etc.), and output devices (e.g., a display device,speakers, and the like). The server and computer may includecomputer-readable media, e.g., memory and storage devices (e.g., flashmemory, hard drive, optical disk drive, magnetic disk drive, and thelike) containing computer instructions that implement the functionalitydisclosed when executed by the processor. The server and the computermay further include wired or wireless network communication interfacesfor communication.

EXAMPLES

The following examples are put forth so as to provide those of ordinaryskill in the art with a complete disclosure and description of how tomake and use the present invention, and are not intended to limit thescope of what the inventors regard as their invention, nor are theyintended to represent or imply that the experiments below are all of orthe only experiments performed. It will be appreciated by personsskilled in the art that numerous variations and/or modifications may bemade to the invention as shown in the specific aspects without departingfrom the spirit or scope of the invention as broadly described. Thepresent aspects are, therefore, to be considered in all respects asillustrative and not restrictive.

Efforts have been made to ensure accuracy with respect to numbers used(e.g., amounts, temperature, etc.) but some experimental errors anddeviations should be accounted for. Unless indicated otherwise, partsare parts by weight, molecular weight is weight average molecularweight, temperature is in degrees centigrade, and pressure is at or nearatmospheric.

Example 1: Subjects

A total of 878 maternal venous blood samples were analyzed with thefollowing classification of trisomy status: 691 were disomic, 18 weretrisomy 13, 37 were trisomy 18, and 132 were trisomy 21. The bloodsamples were obtained from pregnant women at least 18 years old withsingleton pregnancies at 10-34 weeks' gestation. The trisomyclassification had previously been determined for all samples tested;486 samples were originally tested using the Harmony Prenatal Test fromAriosa Diagnostics, Inc. (San Jose, Calif.), and 396 samples weresourced from patients who underwent either amniotic diagnostickaryotyping or postnatal newborn examination followed by karyotypingwhen trisomy was suspected.

Example 2: Sample Preparation

Sample preparation and analysis was performed as described in Sparks, etal., Am J. Obstet Gynecol., 207(5):374.e1-6 (2012). Cell-free DNA waspurified from the plasma of each patient and DANSR™ (Digital Analysis ofSelected Regions) assay products (e.g., tandem ligation products) weremade from loci on chromosomes 13, 18, and 21. For this analysis,ligation assays using sets of fixed sequence oligonucleotides andbridging oligonucleotides corresponding to 864 genomic regions on eachof chromosomes 13, 18, and 21 were used. The DNA sample was attached toa solid support and unhybridized oligonucleotides were removed prior toligation. In addition, ligation assays using sets of fixed sequenceoligonucleotides and bridging oligonucleotides corresponding to 576polymorphic sites on chromosomes 1 through 12 were developed to evaluatethe fraction of fetal cfDNA in each sample. A portion of the ligationassay product produced from each sample was amplified using universalprimers and sequenced and the remaining portion of the ligation assayproduct was hybridized to a custom manufactured DNA array. Prior tohybridization, the amplicons were cleaved. These portions containedidentical products.

Example 3: Ligation Product Quantification Using Arrays

Custom DNA arrays were manufactured by Affymetrix, Inc. (Santa Clara,Calif.) to specifically quantify products of the DANSR assay. DNA arrayswere imaged on an Affymetrix GeneTitan® Multi-Channel (MC) Instrument.Each patient sample was assayed on a single custom DNA array. DNA arrayswere manufactured and processed in interconnected sets of 384. Nextgeneration sequencing data was produced on an Illumina HiSeq® 2500 (SanDiego, Calif.). Clusters were generated on an Illumina Cluster Stationusing TruSeg™ Cluster Generation reagents (San Diego, Calif.). Onaverage 1104 sequencing counts per assay were obtained.

Example 4: Data Analysis

A previously published algorithm entitled Fetal-Fraction Optimized Riskof Trisomy Evaluation (FORTE™), was used to assign risk scores (see,e.g., Sparks, et al., Am J. Obstet Gynecol., 207(5):374.e1-6(2012)).Non-polymorphic ligation assays on chromosomes 13, 18, and 21 were usedto determine chromosome proportion for each of these chromosomes.Polymorphic ligation assays were used to ascertain fetal fraction. Assayvariability was defined as the coefficient of variation (CV) of sequencecounts (sequencing) or intensities (DNA arrays) of an assay acrosssamples; lower assay variability is preferred. Fetal fractionvariability is defined as the relative standard-error of the measuredfetal fraction.

Example 5: Results

For the 878 plasma samples assayed for trisomy risk there was completeconcordance between array-based risk scores and trisomy status using theligation assays and detection by hybridization. In contrast, theconcordance between sequence-based risk scores and trisomy status was99.6%. Sequencing reported high risk scores for two samples that hadreceived low risk scores in a previous NIPT screen. These datademonstrate that trisomy risk scores were accurately obtained fromarray-based analysis.

In addition, array data decreases the assay variability by approximatelytwo-fold.

Accuracy, as measured by decreasing assay variability, was enhanced forarray-based hybridization analysis compared to sequencing-basedanalysis. The median assay variability for array sequence detectionshowed a nearly two-fold improvement overnext-generation-sequencing-based detection (0.051 versus 0.099;p-value<0.0001) (See FIG. 14). The bars of the histogram show the numberof ligation assays (y-axis) that share a specific range of assayvariability (x-axis). Array data is plotted in dark gray, sequencingdata is plotted in white. Where the two populations of data overlap, thebars are light gray. The array-based quantified ligation products havesignificantly lower assay variability, where lower assay variability isbetter.

Arrays can be utilized to lower fetal fraction variability: The fractionof fetal DNA in plasma impacts the accuracy of the testing. The methodsof the present invention measures, reports, and leverages fetal fractionusing the FORTE algorithm to provide highly sensitive results with a lowfalse positive rate (Sparks, et al., Am J Obstet Gynecol.,206(4):319.e1-9 (2012)). FORTE calculated fetal fractions are extremelyreproducible between array-based and sequencing-based analysis data(R{circumflex over ( )}2>0.99). Because the array is able tosimultaneously measure a larger number of ligation assays thanmultiplexed sequencing, fetal fraction estimates using array-basedanalysis are more precise with a median relative standard error of 0.013compared to 0.021.

The data presented in the Examples show that two key sources of datavariability are lower for arrays compared to next generationsequencing: 1) variation of the measured fetal fraction usingpolymorphic assays (fetal fraction variability) and 2) the variation ofnon-polymorphic assays across samples (assay variability). By includingmore polymorphic assays, an array-based ligation product prenatal testhas been engineered that provides lower fetal-fraction variabilityresulting in better precision. By lowering assay variability, smalleraneuploidy changes can be measured, such as the smaller changes observedin low fetal fraction samples. The data demonstrates that the arrayplatform is capable of reliable aneuploidy assessment. Moreover, arrayimaging is a rapid process and the turnaround time for samplequantitation is reduced to less than a minute per sample. The specificdetection system employed in these Examples (GENETITAN™ Multi-ChannelInstrument) can image >90 arrays per machine-hour. In contrast, evenwhen samples are multiplexed in groups of 96 samples per lane, theHISEQ™ 2500 sequencing system has a throughput of 15 samples permachine-hour. This decrease in machine time and complexity translatesdirectly to reductions in capital costs when array sequence detection isused.

The sequence based analysis leverages sample multiplexing in order toachieve economically efficient use of available sequence capacity.However, without normalization, a single sample can consume the majorityof sequence reads in a flow-cell, reducing the reads available fordetermining trisomy risk in the remaining samples. In order to multiplexsamples accurately, laborious and expensive processes to normalizequantities of input DNA are required. Even when efforts are made toequalize sample input, as was reported in a recent study, a four-foldvariation in the median reads per-sample was observed for a 12-plexreaction (Jensen, et al., PLoS ONe, 8(3):e57381 (July 2014). Array-basedNIPT approaches require no sample multiplexing. Instead, each sample ishybridized individually to a single array. Processing throughput isenhanced by physically connecting, e.g., 384 arrays onto a singlemulti-array plate for convenient high throughput handling. Because eachsample is handled individually, time is saved and cost is reduced.

While this invention is satisfied by aspects in many different forms, asdescribed in detail in connection with preferred aspects of theinvention, it is understood that the present disclosure is to beconsidered as exemplary of the principles of the invention and is notintended to limit the invention to the specific aspects illustrated anddescribed herein. Numerous variations may be made by persons skilled inthe art without departure from the spirit of the invention. The scope ofthe invention will be measured by the appended claims and theirequivalents. The abstract and the title are not to be construed aslimiting the scope of the present invention, as their purpose is toenable the appropriate authorities, as well as the general public, toquickly determine the general nature of the invention. In the claimsthat follow, unless the term “means” is used, none of the features orelements recited therein should be construed as means-plus-functionlimitations pursuant to 35 U.S.C. § 112, ¶16.

1-20. (canceled)
 21. An assay method for determining the fraction offetal DNA in a sample, comprising the steps of: (a) providing a maternalplasma or serum sample comprising maternal and fetal cell free DNA; (b)interrogating at least 48 polymorphic loci from at least one targetgenomic region in the maternal plasma or serum sample by hybridizing aset of at least two fixed sequence oligonucleotides for each allele ateach polymorphic locus to the maternal and fetal cell free DNA, whereinone of the at least two fixed sequence oligonucleotides of each setcomprises a sequence complementary to one allele at a polymorphic locus,a capture region specific for each polymorphic locus, an allele-specificlabel binding region, and two restriction sites; (c) ligating each setof hybridized fixed sequence oligonucleotides; (d) amplifying theligated fixed sequence oligonucleotides to create allele-specificamplicons; (e) cleaving the allele-specific amplicons at the restrictionsites to create cleaved allele-specific amplicons, wherein each cleavedallele-specific amplicon comprises a polymorphic locus-specific captureregion and an allele-specific label binding region; (f) detecting thecleaved allele-specific amplicons from the polymorphic loci viacompetitive hybridization of the polymorphic locus-specific captureregions of the cleaved allele-specific amplicons to capture probes on anarray; and (g) quantifying the alleles of the polymorphic loci bydetecting the allele-specific label binding regions for each allele onthe cleaved allele-specific amplicons on the array, (g) therebydetermining the fraction of fetal DNA in the sample.
 22. The assaymethod of claim 21, wherein three or more fixed sequenceoligonucleotides for set are used to interrogate each polymorphic locus.23. The assay method of claim 21, wherein the amplifying step comprisesuniversal amplification through a polymerase chain reaction.
 24. Theassay method of claim 21, wherein the at least one target genomic regioncomprises at least one chromosome.
 25. The assay method of claim 21,wherein the at least one target genomic region comprises two or morechromosomes.
 26. The assay method of claim 24, wherein the at least onechromosome is not chromosome 13, 18, or
 21. 27. The assay method ofclaim 21, wherein the allele-specific label binding regions eachcomprise a label specific for one allele at a polymorphic locus.
 28. Theassay method of claim 21, wherein the allele-specific label bindingregions each comprise a sequence complementary to an allele-specificoligonucleotide comprising an allele-specific label.
 29. An assay methodfor determining the fraction of fetal DNA in a sample, comprising thesteps of: (a) providing a maternal plasma or serum sample comprisingmaternal and fetal cell free DNA; (b) interrogating at least 48polymorphic loci from at least one target genomic region in the maternalplasma or serum sample by hybridizing a set of at least two fixedsequence oligonucleotides for each allele at each polymorphic locus tothe maternal and fetal cell free DNA, wherein at least two of the fixedsequence oligonucleotides of each set comprise a universal primer site,and wherein at least one of the fixed sequence oligonucleotides of eachset comprises a sequence complementary to one allele at a polymorphiclocus, a capture region specific for each polymorphic locus, anallele-specific label binding region, and two restriction sites; (c)extending at least one of the hybridized fixed sequence oligonucleotidesof each set to form adjacently hybridized fixed sequenceoligonucleotides. (c) ligating each set of hybridized fixed sequenceoligonucleotides; (d) amplifying the ligated fixed sequenceoligonucleotides using the universal primer sites to createallele-specific amplicons; (e) cleaving the allele-specific amplicons atthe restriction sites to create cleaved allele-specific amplicons,wherein each cleaved allele-specific amplicon comprises a polymorphiclocus-specific capture region and an allele-specific label bindingregion; (f) applying the cleaved amplicons to an array, wherein thearray comprises allele-specific capture probes complementary to theallele-specific capture regions of the cleaved amplicons; (g)hybridizing the allele-specific capture regions of the cleaved ampliconsto the allele-specific capture probes on the array; (h) detecting thehybridized cleaved amplicons; (i) quantifying the relative frequency ofcleaved amplicons corresponding to each allele of each polymorphic locusby detecting each allele-specific label binding region; and (j)calculating the fraction of fetal DNA in the sample using the relativefrequency of cleaved amplicons corresponding to each allele of eachpolymorphic locus.
 30. The assay method of claim 29, wherein a singlehybridized fixed sequence oligonucleotide of each set is extended toform adjacently hybridized fixed sequence oligonucleotides.
 31. Theassay method of claim 29, wherein three or more fixed sequenceoligonucleotides for each set are used to interrogate each polymorphiclocus.
 32. The assay method of claim 29, wherein the amplifying stepcomprises universal amplification through a polymerase chain reaction.33. The assay method of claim 29, wherein the at least one targetgenomic region comprises at least one chromosome.
 34. The assay methodof claim 29, wherein the at least one target genomic region comprisestwo or more chromosomes.
 35. The assay method of claim 33, wherein theat least one chromosome is not chromosome 13, 18, or
 21. 36. The assaymethod of claim 29, wherein the allele-specific label binding regionseach comprise a label specific for one allele at a polymorphic locus.37. The assay method of claim 21, wherein the allele-specific labelbinding regions each comprise a sequence complementary to anallele-specific oligonucleotide comprising an allele-specific label. 38.An assay method for determining the fraction of fetal DNA in a sample,comprising the steps of: (a) providing a maternal plasma or serum samplecomprising maternal and fetal cell free DNA; (b) interrogating at least48 polymorphic loci from at least one target genomic region in thematernal plasma or serum sample by hybridizing a set of at least threefixed sequence oligonucleotides for each allele at each polymorphiclocus to the maternal and fetal cell free DNA, wherein at least two ofthe fixed sequence oligonucleotides of each set comprises a universalprimer site, and wherein at least one of the fixed sequenceoligonucleotides of each set comprises a sequence complementary to oneallele at a polymorphic locus, a capture region specific for eachpolymorphic locus, an allele-specific label binding region, and tworestriction sites; (c) ligating each set of hybridized fixed sequenceoligonucleotides; (d) amplifying the ligated fixed sequenceoligonucleotides to create allele-specific amplicons; (e) cleaving theallele-specific amplicons at the restriction sites to create cleavedallele-specific amplicons, wherein each cleaved allele-specific ampliconcomprises a polymorphic locus-specific capture region and anallele-specific label binding region; (f) applying the cleaved ampliconsto an array, wherein the array comprises allele-specific capture probescomplementary to the allele-specific capture regions of the cleavedamplicons; (g) hybridizing the allele-specific capture regions of thecleaved amplicons to the allele-specific capture probes on the array;(h) detecting the hybridized cleaved amplicons; (i) quantifying therelative frequency of cleaved amplicons corresponding to each allele ofeach polymorphic locus by detecting each allele-specific label bindingregion; and (j) calculating the fraction of fetal DNA in the sampleusing the relative frequency of cleaved amplicons corresponding to eachallele of each polymorphic locus.
 39. The assay method of claim 38,wherein the amplifying step comprises universal amplification through apolymerase chain reaction.
 40. The assay method of claim 38, wherein theat least one target genomic region comprises at least one chromosome.41. The assay method of claim 38, wherein the at least one targetgenomic region comprises two or more chromosomes.
 42. The assay methodof claim 40, wherein the at least one chromosome is not chromosome 13,18, or
 21. 43. The assay method of claim 38, wherein the allele-specificlabel binding regions each comprise a label specific for one allele at apolymorphic locus.
 44. The assay method of claim 38, wherein theallele-specific label binding regions each comprise a sequencecomplementary to an allele-specific oligonucleotide comprising anallele-specific label.